Recombinant bacterial cells for delivery of PNP to tumor cells

ABSTRACT

The present invention provides a procaryotic host cell stably transformed or transfected by a vector including a DNA sequence encoding for purine nucleoside phosphorylase or hydrolase. The transformed or transfected procaryotic host cell can be used in combination with a purine substrate to treat tumor cells and/or virally infected cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 09/183,188filed Oct. 30, 1998, now U.S. Pat. No. 6,491,905 which is anon-provisional of provisional Ser. No. 60/064,676 filed Oct. 31, 1997,and is also a continuation-in-part application of U.S. Ser. No.08/881,772 filed Jun. 24, 1997, now U.S. Pat. No. 6,017,896, which is acontinuation-in-part application of U.S. Ser. No. 08/702,181 filed Aug.23, 1996, now abandoned, which is a continuation-in-part application ofU.S. Ser. No. 08/122,321 filed Sep. 14, 1993, now U.S. Pat. No.5,552,311.

GRANT REFERENCE

The research carried out in connection with this invention was supportedin part by a grant from the National Cancer Institute (CA 7763-02).

SEQUENCE LISTING

This application contains a Sequence Listing that is being submittedherewith as a separate document.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of cancer therapy and in particular,relates to compositions and methods to specifically kill tumor cells bythe production of toxic compounds in the tumor cells.

2. Description of the Related Art

Inefficiency of gene delivery, together with inadequate bystanderkilling, represent two major conceptual hurdles in the development of atoxin mediated gene therapy for human malignancy. Gene transfer is auseful adjunct in the development of new therapies for human malignancy.Tumor cell expression of histocompatibility antigens, cytokines, orgrowth factors (for example, IL-2, IL-4, GMCSF) appears to enhanceimmune-mediated clearance of malignant cells in animal models, andexpression of chemo-protectant gene products, such as p-glycoprotein inautologous bone marrow cells, is under study as a means of minimizingmarrow toxicity following administration of otherwise lethal doses ofchemotherapeutic agents.

Theoretically, the most direct mechanism for tumor cell killing usinggene transfer is the selective expression of cytotoxic gene productswithin tumor cells. However, no recombinant enzyme or toxin has provenuseful in mediating high levels of toxicity in unselected tumor cells.Classical enzymatic toxins such as pseudomonas exotoxin A, diphtheriatoxin and ricin are unlikely to be useful in this context, since theseenzymes kill only cells in which they are expressed, and no currentlyavailable gene transfer vector is capable of gene delivery to asufficiently high percentage of tumor cells to make use of the aboverecombinant enzymes.

Another strategy that has been developed to selectively kill tumor cellsinvolves the delivery and expression of the HSV dThd kinase gene toreplicating tumor cells followed by treatment with ganciclovir.Ganciclovir is readily phosphorylated by the HSV dThd kinase, and itsphosphorylated metabolites are toxic to the cell. Very littlephosphorylation of ganciclovir occurs in normal human cells. Althoughonly those cells expressing the HSV dThd kinase should be sensitive toganciclovir (since its phosphorylated metabolites do not readily crosscell membranes), in vitro and in vivo experiments have shown that agreater number of tumor cells are killed by ganciclovir treatment thanwould be expected based on the percentage of cells containing the HSVdThd kinase gene. This unexpected result has been termed the “bystandereffect” or “metabolic cooperation.” It is thought that thephosphorylated metabolites of ganciclovir may be passed from one cell toanother through gap junctions. However, even if a nucleosidemonophosphate such as ganciclovir monophosphate were released into themedium by cell lysis, the metabolite would not be able to enterneighboring cells and would likely be degraded (inactivated) to thenucleoside by phosphatases.

Although the bystander effect has been observed in initial experimentsusing HSV dThd kinase, the limitations of current gene delivery vehiclesmean that a much greater bystander effect is important to successfullytreat human tumors using this approach. One difficulty with the currentbystander toxicity models is that bystander toxicity with metabolitesthat do not readily cross the cell membrane will not be sufficient toovercome a low efficiency of gene transfer (for example, transfection,transduction, etc.).

One protocol for treating brain tumors in humans uses retroviraldelivery of HSV dThd kinase, followed by ganciclovir administration. Inrat models, using HSV dThd in this context, tumor regressions have beenobserved. The HSV dThd kinase approach has not proven sufficient inhumans thus far; this may in part be due to (1) inadequate bystandertoxicity with HSV dThd kinase, and (2) cell killing only of dividingcells using HSV dThd kinase with ganciclovir. The usefulness of E. colicytosine deaminase, which converts 5-fluorocytosine to 5-fluorouracil,has recently been reported to provide substantial bystander toxicity.However, 5-FU is not a highly toxic compound in this setting andbystander killing in vitro has been inefficient, i.e., similar to thatof observed with HSV dThd kinase.

Prodrug activation by an otherwise non-toxic enzyme (for example, HSVdThd kinase, cytosine deaminase) has advantages over the expression ofdirectly toxic genes, such as ricin, diphtheria toxin, or pseudomonasexotoxin. These advantages include the capability to (1) titrate cellkilling, (2) optimize therapeutic index by adjusting either levels ofprodrug or of recombinant enzyme expression, and (3) interrupt toxicityby omitting administration of the prodrug. However, like otherrecombinant toxic genes, gene transfer of HSV dThd kinase followed bytreatment with ganciclovir is neither designed to kill bystander cellsnor likely to have broad bystander toxicity in vivo.

An additional problem with the use of the HSV dThd kinase or cytosinedeaminase to create toxic metabolites in tumor cells is the fact thatthe agents activated by HSV dThd kinase (ganciclovir, etc.) and cytosinedeaminase (5-fluorocytosine) kill only cells synthesizing DNA. Even if aconsiderable number of nontransfected cells are killed, one would notexpect to kill the nondividing tumor cells with these agents.

Thus, there exists a need for a toxin gene therapy method that overcomesthe problems of inefficient gene delivery, cell replication-dependentkilling and low toxin diffusion between cells. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

Accordingly to the present invention, a unique E. coli containing thePNP gene (SEQ ID No:5) is disclosed. This E. coli can be used to treattumors in combination with a prodrug including MeP-dR. Also, a methodfor causing tumor regression and/or inhibiting tumor growth is disclosedwhich includes directly administering a purine analog to a tumor.

In yet still another embodiment of the present invention, there isprovided a host cell transfected with the vector of the presentinvention which expresses a purine nucleoside phosphorylase protein.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the toxicity due to DOTMA-DOPE liposomes used to transfectT-84 colon carcinoma cells with 10, 20 or 40 μg of cDNA containingeither the E. coli PNP or LacZ genes under the transcriptional controlof SV-40 early promoter (SV-PNP and SV-LacZ, respectively) and theadditional toxicity when MeP-dR (160 μM) is added to T-84 transfectedcells expressing the PNP gene (PNP+MeP-dR). Cells transfected withSV-PNP construct were treated, with (PNP+MeP-dR) and without (PNP)MeP-dR. LacZ transfected cells were studied in the same way.Nontransfected cells were treated with (MeP-dR) and without (control)MeP-dR.

FIGS. 2A-D show the human tyrosinase transcriptional promoter sequence(Tyr)-restricted expression of the luciferase reporter gene (Luc), towhich it was operable linked (Tyr-Luc), in melanoma cells Mel-1 andMel-21 (FIG. 2A), and the SV40 early promoter (SV) constitutiveexpression of the Luciferase gene (Luc) to which it was operable linked(SV-Luc), in each carcinoma cell line (see FIGS. 2A-2D). Rev-Tyr-Luc,Tyr promoter sequence linked to the Luc gene in reverse orientation sothat it does not transcribe Luc (no expression). Basic, promoterless Lucgene construct.

FIGS. 3A and 3B show the dependence of purine analog nucleoside MeP-dRtoxicity on expression of E. coli purine analog nucleoside phosphorylase(PNP). SV-PNP, cells transfected with a construct in which theconstitutive SV40 early promoter is operably linked to the PNP gene;Tyr-PNP, cells transfected with a construct in which the melanomaspecific human tyrosinase promoter sequence is operably linked to PNPgene; Tyr-Luc, cells transfected with a construct in which the melanomaspecific human tyrosinase promoter sequence is operably linked toluciferase reporter gene; no-txf, cells not transfected with arecombinant construct. T-84, carcinoma cell line (3A); Mel-1, melanomacell line (3B).

FIG. 4 shows the difference in in vivo development of tumors in athymicnude mice engrafted with murine mammary carcinoma 16/C cells transducedwith the recombinant retroviral expression vector LN/PNP (which directsexpression of E. coli PNP) depending on time of administration of MeP-dRprodrug. No injection of MeP-dR (control); injection of MeP-dR on days1-4 post engraftment (early rx); injection of MeP-dR on days 13-15 postengraftment (late rx) are shown.

FIG. 5 shows the effect of MeP-dR on transduced cells with stable E.coli PNP expression. FIGS. 5A and 5B show mixing experiments in whichthe transduced and wild type B16 (FIG. 5A) or 16/C (FIG. 5B) werecocultured. Complete abrogation of cellular proliferation was observedwhen as few as 2% of the cultured cells expressed E. coli PNP under theregulatory control of an SV-40 promoter. A high level bystander effectwas also observed when either B16 or 16/C cells expressed E. coli PNP,as measured by a standard cellular LDH release assay. Growthcharacteristics of transduced and wild type (nontransduced) B16 cellswere identical in the absence of drug; the same was true of the wildtype and transduced 16/C cell lines.

FIG. 6 shows the effect of MeP-dR and F-araAMP on the growth ofwild-type 16/C tumors in animals. Both compounds had only a small effecton tumor growth. These results are in contrast with those in FIGS. 4 and7.

FIG. 7 shows the effect of F-araAMP on the growth of 16/C tumorsexpressing E. coli PNP. F-araAMP significantly inhibited the growth ofthese tumors. Contrast with the effect of F-araAMP on wild-type tumorsin FIG. 6.

FIGS. 8 and 9 show the effect of MeP-dR on the growth of wild-type D54tumors (FIG. 8) and E. coli PNP expressing D54 tumors (FIG. 9). Thesetwo figures are a graphical representation of the data shown in TableIV. FIG. 8 shows that MeP-dR did not affect parental D54 tumor cellgrowth. FIG. 9 shows that MeP-dR caused regression of D54 tumorsexpressing E. coli PNP. Note that in this figure tumors that completelyregressed are not included in the calculation of medium tumor weight.Therefore, since 4 animals had no tumors at the end of the experiment,the tumor weight on the days beyond day 40 refer to the two tumors thatdid not completely regress. In this experiment the two remaining tumorswere at the limit of detection and did not show any signs of growth pastday 30. Therefore, these animals may also be cured of their disease.

FIGS. 10 and 11 are a confirmation study of the experiment shown inFIGS. 8 and 9 that show the effect of MeP-dR on the growth of wild-typeD54 tumors (FIG. 10) and E. coli PNP expressing D54 tumors (FIG. 11).These two figures are a graphical representation of the data shown inTable V. FIG. 10 shows that MeP-dR at two doses did not affect parentalD54 tumor cell growth. FIG. 11 shows that MeP-dR at both doses causedregression of D54 tumors expressing E. coli PNP. Note that as in FIG. 9,tumors that completely regressed are not included in the calculation ofmedium tumor weight. Therefore, since 4 animals, which were treated with67 mg/kg MeP-dR, had no tumor at the end of the experiment, the tumorweight on the days beyond day 40 refer to the six tumors that did notcompletely regress.

FIG. 12 is a predicted restriction map of plasmid pTRCPNP containing thepredicted 5013 base sequence pairs (SEQ ID No: 5) encoding E. coli PNP.

FIG. 13 illustrates the induction of E. coli PNP by IPTG.

FIG. 14 is a graph of median tumor weight versus the response of SCLewis Lung Tumors to treatment with MeP-dR and NSC 103543.

FIG. 15 is a histogram illustrating the tumor burden versus the tumorvolume on day 16 following treatment with MeP.

FIG. 16 is a graph illustrating tumor burden over time followingtreatment with MeP.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of killing replicating ornon-replicating, transfected or transduced mammalian cells and bystandercells, comprising the following steps: (a) transfecting or transducingtargeted mammalian cells with a nucleic acid encoding a suitable purinenucleoside cleavage enzyme which releases a purine analog from thesubstrate purine nucleoside or providing such enzyme directly to thetargeted cells; and (b) contacting the targeted cells expressing orprovided with the purine nucleoside cleavage enzyme with a substrate forthe enzyme to produce a toxic purine base thereby killing the targetedcells and also bystander cells not expressing or containing the cleavageenzyme. Thus, in the presence of substrate, the cleavage enzyme producesa toxic product. It should be appreciated that a “non-human or modifiedhuman purine analog nucleoside phosphorylase (PNP)” includes the use ofeither types of PNP in the same therapeutic regimen as the purinenucleoside cleavage enzyme. The killing can occur in vitro or in vivo.

In this method of the present invention, the targeted cells arepreferably selected from the group consisting of tumor cells and virallyinfected cells. In one suitable instance, the natural or modified enzymeis a non-human PNP or hydrolase. More preferably, the hydrolase is anucleoside hydrolase. Alternatively, the enzyme is a modified mammalianPNP or hydrolase. PNP includes subgroups such as the MTAP(methylthioadenosine phosphorylase).

In one embodiment of this method of the present invention, the enzyme isprovided by targeting the enzyme to the cells. More preferably, theenzyme is targeted to the cells by conjugating the enzyme to anantibody.

The enzyme may be encoded by a gene provided to the cells. For example,the gene provided to the cells encodes E. coli PNP and is operablylinked to a tyrosinase gene promoter. Alternatively, the gene isprovided in a carrier molecule such as polymeric films, gels,microparticles and liposomes.

In another embodiment, the present invention provides a method ofkilling replicating or non-replicating, targeted mammalian cells andbystander cells, comprising the following steps: (a) delivering a purinenucleoside phosphorylase to the targeted mammalian cells; and (b)contacting the targeted cells with an effective amount of a nucleosidesubstrate for the purine nucleoside phosphorylase, wherein the substrateis non-toxic to mammalian cells and is cleaved by the phosphorylase toyield a purine base which is toxic to the targeted mammalian cells andbystander cells, to kill the mammalian cells expressing thephosphorylase and the bystander cells. Representative examples of purineanalog substrates include9-(β-D-2-deoxyerythropentofuranosyl)-6-methypurine,2-amino-6-chloro-1-deazapurine riboside, 7-ribosyl-3-deazaguanine,arabinofuranosyl-2-fluomadenine, 2-fluoro-2′-deoxyadenosine,2-fluoro-5′-deoxyadenosine, 2-chloro-2′-deoxyadenosine,5′-amino-5′-deoxyadenosine, α-adenosine, MeP-2′,3′-dideoxyriboside,2-F-2′,3′-dideoxyadenosine, MeP-3′-deoxyriboside, 2-F-3′-deoxyadenosine,2-F-adenine-6′-deoxy-β-D-allofuranoside, 2-F-adenine-α-L-lyxofuranoside,MeP-6′-deoxy-β-D-allofuranoside, MeP-α-L-lyxofuranoside,2-F-adenine-6′-deoxy-α-L-talofuranoside,MeP-6′-deoxy-α-L-talofuranoside.

The present invention also provides a composition for killing targetedmammalian cells, comprising: (a) an enzyme that cleaves a purinesubstrate; and (b) an effective amount of the purine analog substrate tokill the targeted cells when cleaved by the enzyme.

The present invention is also directed to a vector comprising a DNAsequence coding for a purine nucleoside phosphorylase protein and saidvector is capable of replication in a host which comprises, in operablelinkage: a) an origin of replication; b) a promoter; and c) a DNAsequence coding for said protein. Preferably, the vector is selectedfrom the group consisting of a retroviral vector, an adenoviral vector,an adeno-associated viral vector, a herpes vector, a viral vector and aplasmid.

The present invention also includes a method for inhibiting tumor growthby directly administering to a tumor a purine analog or derivativethereof.

The present invention is also directed to a host cell transfected withthe vector of the present invention so that the vector expresses an E.coli purine nucleoside phosphorylase protein. Preferably, such hostcells are selected from the group consisting of bacterial cells,mammalian cells and insect cells.

Some of the methods and compositions, exemplified below, involvetransfecting cells with the E. coli DeoD gene (encoding a purine analognucleoside phosphorylase (PNP)) and subsequently treating with anontoxic purine nucleoside, e.g., deoxyadenosine or deoxyguanosineanalogs, including N7 analogs), which is converted to a toxic purineanalog. E. coli PNP differs from human PNP in its more efficientacceptance of adenine and certain guanine-containing nucleoside analogsas substrates. E. coli PNP expressed in tumor cells cleaves thenucleoside, liberating a toxic purine analog. Purine analogs freelydiffuse across cell membranes, whereas nucleoside monophosphates such asthose generated using HSV Thd kinase, generally remain inside the cellin which they are formed. A toxic adenine analog formed after conversionby E. coli PNP can be converted by adenine phosphoribosyl transferase totoxic nucleotides and kill all transfected cells, and diffuse out of thecell and kill surrounding cells that were not transfected (bystandercells).

Enzymes Catalyzing Purine Analog Conversion

Two classes of enzymes can be used: phosphorylases and nucleosidasehydrolases. A PNP useful in the methods and compositions describedherein catalyzes the conversion of purine analog nucleosides plusinorganic phosphate to free the toxic purine analog plusribose-1-phosphate (or deoxyribose-1-phosphate): purine analognucleoside+PO₄: purine analog ribose-1-PO₄ (ordeoxyribose-1-phosphate)+toxic purine analog. Methylthioadenosinephosphorylase, a subclass of PNP, would also be useful in this context.Non-mammalian and modified human or modified other mammalian PNPs can beused. The non-mammalian PNP can be an E. coli purine analog nucleosidephosphorylase. However, any PNP which can selectively convert asubstrate to produce a toxic purine analog can be utilized. Thus,modifications in the E. coli PNP, which retain this activity, are withinthe scope of the class of enzymes suitable for the described methods andcompositions, as are human PNP enzyme molecules that have been modifiedto cleave purine analog nucleoside to release the toxic purine analogmoiety. A method is presented below by which any proposed PNP or otherpurine analog nucleoside cleavage enzyme can be tested in a cell for itsability to convert a given substrate from a relatively nontoxic form toa toxin for the cells.

Table I lists organisms which possess an enzyme that cleavesadenine-containing nucleosides to adenine and so are useful in themethods described herein. Table I also shows that humans and the malariaparasite Plasmodium falciparum do not possess an enzyme useful in thedescribed methods. Thus, to be useful in the methods described herein, ahuman or P. falciparum PNP would have to be modified torbe capable ofcleaving a purine analog nucleoside substrate to liberate this toxicpurine analog. Such modifications can be made at the genetic level orprotein level. For example, in vitro mutagenesis of the gene encodingthe human or P. falciparum PNP can be used to alter the gene sequence toencode a PNP that will cleave a particular purine analog nucleoside.

As described above, in a preferred embodiment, the PNP used in thepresent methods can include genetically modified mammalian ornon-mammalian PNP, as well as bacterial PNP, capable of reacting with asubstrate that the native PNP in the tumor cell will not recognize orrecognizes poorly. Thus, the nucleic acids that encode a useful PNP arepresent in cells in which they are not naturally found, either becausethey are from a different organism or because they have been modifiedfrom their natural state. The key requirement of the nucleic acidsencoding the PNP or other purine analog nucleoside cleavage enzyme isthat they must encode a functional enzyme that is able to recognize andact upon a substrate that is not well recognized by the native PNP ofthe cell.

Nucleosidases or nucleoside hydrolases are another class of enzymessuitable for the methods and compositions described herein. Thedefinition of a purine analog nucleosidase is an enzyme that catalyzesthe conversion of purine analog nucleosides plus water to liberate freetoxic purine analogs plus ribose (or deoxyribose): purine analognucleoside+H₂O: purine analog+ribose (or deoxyribose).

Transcriptional Regulation of the PNP Encoding Sequence

Since a bacterial PNP is encoded on a prokaryotic gene, the expressionof the bacterial PNP in mammalian cells will require a eukaryotictranscriptional regulatory sequence linked to the PNP-encodingsequences. The bacterial PNP gene can be expressed under the control ofstrong constitutive promoter/enhancer elements that are obtained withincommercial plasmids (for example, the SV40 early promoter/enhancer(pSVK30 Pharmacia, Piscataway, N.J., cat. no. 27-4511-01), moloneymurine sarcoma virus long terminal repeat (pBPV, Pharmacia, cat. no.4724390-01), mouse mammary tumor virus long terminal repeat (pMSG,Pharmacia, cat. no. 27-4506-01), and the cytomegalovirus earlypromoter/enhancer (pCMVβ, Clontech, Palo Alto, Calif., cat. no.6177-1)).

Selected populations of cells can also be targeted for destruction byusing genetic transcription regulatory sequences that restrictexpression of the bacterial PNP (or other suitable purine analognucleoside cleavage enzyme) coding sequence to certain cell types, astrategy that is referred to as “transcription targeting. ” A candidateregulatory sequence for transcription targeting must fulfill twoimportant criteria as established by experimentation: (i) the regulatorysequence must direct enough gene expression to result in the productionof enzyme in therapeutic amounts in targeted cells, and (ii) theregulatory sequence must not direct the production of sufficient amountsof enzyme in non-targeted cells to impair the therapeutic approach. Inthis form of targeting, the regulatory sequences are functionally linkedwith the PNP sequences to produce a gene that will only be activated inthose cells that express the gene from which the regulatory sequenceswere derived. Regulatory sequences that have been shown to fulfill thecriteria for transcription targeting in gene therapy include regulatorysequences from the secretory leucoprotease inhibitor, surfactant proteinA, and α-fetoprotein genes. A variation on this strategy is to utilizeregulatory sequences that confer “inducibility” so that localadministration of the inducer leads to local gene expression. As oneexample of this strategy, radiation-induced sequences have beendescribed and advocated for gene therapy applications. It is expectedthat bacterial PNP gene expression could be targeted to specific sitesby other inducible regulatory elements.

It may be necessary to utilize tissue-specific enhancer/promoters as ameans of directing PNP expression, and thereby PNP-mediated toxicity, tospecific tissues. For example, human tyrosinase genetic regulatorysequences are sufficient to direct PNP toxicity to malignant melanomacells. Mouse tyrosinase sequences from the 50 flanking region (−769 bpfrom the transcriptional start site) of the gene were capable ofdirecting reporter gene expression to malignant melanoma cells. Althoughthe mouse and human tyrosinase sequences in the 50 flanking region aresimilar, Shibata et al., Journal of Biological Chemistry,267:20584-20588 (1992) have shown that the human 50 flanking sequencesin the same region used by Vile and Hart (−616 bp from thetranscriptional start site) did not confer tissue specific expression.Although Shibata et al. suggested that the 50 flanking region would notbe useful to target gene expression to tyrosinase expressing cells(melanomas or melanocytes), a slightly different upstream fragment fromthat used by Shibata et al., can in fact direct reporter or bacterialPNP gene expression specifically to melanoma cells, as shown in FIG. 3.

The 50 flanking region of the human tyrosinase gene was amplified by thepolymerase chain reaction from human genomic DNA. The primers weredesigned to amplify a 529 bp fragment that extended −451 to +78 bprelative to the transcription start site by using a published sequenceof the human tyrosinase gene and flanks (Kikuchi, et al., Biochimica etBiophysica Acta, 1009:283-286 (1989)). The fragment was shown byreporter gene assays to be able to direct reporter gene expression inmelanoma cells (FIG. 2). The same tyrosinase fragment was used to directPNP expression within a plasmid vector and shown to result in PNPmediated toxicity only in melanoma cells (FIG. 3). Therefore, humantyrosinase sequences are useful to direct PNP expression to humanmelanoma cells. These same sequences could be useful to direct othertherapeutic gene expression in melanoma cells or melanocytes. Othertissue-specific genetic regulatory sequences and elements can be used todirect expression of a gene encoding a suitable purine analog nucleosidecleavage enzyme to specific cell types other than melanomas.

TABLE I Organism Enzyme Organisms which can cleave adenine-containingnucleosides to adenine Leishmania donvani Hydrolase Trichomomasvaginalis Phosphorylase Trypanosoma cruzi Hydrolase Schistosoma mansoniPhosphorylase Leishmania tropica Hydrolase Crithidia FasciculataHydrolase Aspergilis and Penicillium Hydrolase Erwinia carotovoraPhosphorylase Helix pomatia Phosphorylase Ophiodon elongatus (lingcod)Phosphorylase E. coli Phosphorylase Salmonella typhimurium PhosphorylaseBacillus subtilis Phosphorylase Clostridium Phosphorylase mycoplasmaPhosphorylase Trypanosoma gambiense Hydrolase Trypanosoma bruceiPhosphorylase (methylthio adenosine phosphorylase) Organisms whichcannot (or poorly) convert adenine-containing nucleosides to adenineHuman Phosphorylase P. falciparum PhosphorylaseSubstrate Selection

A purine analog nucleoside which is a substrate for the enzyme toproduce a toxic substance which kills the cells is referred to herein asa “prodrug.” Any deoxypurine analog nucleoside composed of the cytotoxicpurine bases including those listed below and in Table II should be asubstrate for the E. coli PNP or other equivalent purine analognucleoside cleavage enzyme. A requisite is that the analog must have alow toxicity at the nucleoside level (that is, as a prodrug). Usingribose- or deoxyribose-containing substrates, E. coli PNP canselectively produce a variety of toxic guanine analogs, such as6-thioguanine or 3-deazaguanine, that are attached to ribose ordeoxyribose via the N-7 position in the guanine ring. The strategydescribed here for therapeutic PNP gene transfer implicates new uses forseveral broad classes of specifically activatable cytotoxic purineanalogs in the treatment of human malignancy. Because the growthfraction is very small in most tumors, it is sometimes preferable toselect compounds that are active against both dividing and nondividingcells. Some of the toxic purine analogs produced using E. coli PNP inthe present method are toxic to nondividing as well as dividing cells.Specific examples of suitable purine analog nucleosides that will workin the compositions and methods described herein can be tested accordingto the protocols set forth in the Examples.

In a preferred embodiment described in the Examples, the substrate is9-(β-D-2-deoxyerythropentofuranosyl)-6-methylpurine (MeP-dR). AlthoughMeP-dR is relatively non-toxic, the therapeutic index of this compoundcan be enhanced. For instance, if the toxicity of MeP-dR is due tophosphorylation by a deoxynucleoside kinase, then analogs that cannot bephosphorylated, such as 50-deoxy-MeP-dR, can be synthesized and used asthe prodrug to generate MeP in vivo.

The compounds 6-methylpurine-20-deoxyriboside (Gene Therapy, 1:233-238,1994), 2-amino-6-chloro-1-deazapurine riboside (Biochem. Pharmacol.,33:261-271, 1984), and 7-ribosyl-3-deazaguanine (Biochem. Pharmacol.,29:1791-1787, 1979) are examples of prodrugs that are useful substratesfor the E. coli PNP. They are much less toxic than their respectivepurine analogs.

Delivery of the PNP Gene

Described below is the construction of suitable recombinant viruses andthe use of adenovirus for the transfer of bacterial PNP into mammaliancells. Non-viral gene delivery can also be used. Examples includediffusion of DNA in the absence of any carriers or stabilizers (“nakedDNA”), DNA in the presence of pharmacologic stabilizers or carriers(“formulated DNA”), DNA complexed to proteins that facilitate entry intothe cell (“Molecular conjugates”), or DNA complexed to lipids. The useof lipid-mediated delivery of the bacterial PNP gene to mammalian cellsis exemplified below. More particularly, cationic liposome-mediatedtransfer of a plasmid containing a non-human PNP gene is demonstrated.However, other gene transfer methods will also generally be applicablebecause the particular method for transferring the PNP gene to a cell isnot solely determinative of successful tumor cell killing. Thus, genetransduction, utilizing a virus-derived transfer vector, furtherdescribed below, can also be used. Such methods are well known andreadily adaptable for use in the gene-mediated toxin therapies describedherein. Further, these methods can be used to target certain diseasesand cell populations by using the targeting characteristics of aparticular carrier of the gene encoding a suitable purine analognucleoside cleavage enzyme such as E. coli PNP.

Apathogenic anaerobic bacteria have been used to selectively deliverforeign genes into tumor cells. For example, Clostridium acetobutylicumspores injected intravenously into mice bearing tumors, germinated onlyin the necrotic areas of tumors that had low oxygen tension. Using thestandard PNP assay described below, Clostridium perfringens (SigmaChemical Co., St. Louis, Mo.) was found to exhibit enzyme activitycapable of converting MeP-dR to MeP. This finding suggests a mechanismto selectively express bacterial PNP activity in tumor masses withnecrotic, anaerobic centers. Thus, tumors can be infected with suchstrains of Clostridium and then exposed to a purine analog such asMeP-dR. The PNP activity of the clostridium bacteria growing in theanaerobic center of the tumor tissue should then convert the MeP-dR toMeP, which then is released locally to kill the-tumor cells.Additionally, other bacteria including E. coli and Salmonella can beused to deliver the PNP gene or hydrolase into tumors. As described anddemonstrated below in Example 25, E. coli containing a plasmid (see FIG.12) encoding the E. coli PNP gene plus MeP-dR demonstrated efficaciousanti-tumor activity (slowing of tumor growth) in mice and also thatdelivery of significant amounts of E. coli PNP to tumor cells in animalscould activate MeP-dR resulting in anti-tumor response.

The rapidly advancing field of therapeutic DNA delivery and DNAtargeting also includes vehicles such as “stealth” and otherantibody-conjugated liposomes (including lipid-mediated drug targetingto colonic carcinoma), receptor-mediated targeting of DNA through cellspecific ligands, lymphocyte-directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells in vivo(S. K. Huang et al., Cancer Research, 52:6774-6781 (1992); R. J. Debs etal., Am. Rev. Respir. Dis., 135:731-737 (1987); K. Maruyama et al.,Proc. Natl. Acad. Sci. USA, 87:5744-5748 (1990); P. Pinnaduwage and L.Huang, Biochemistry, 31:2850-2855 (1992); A. Gabizon andPapahadjopoulas, Proc. Natl. Acad. Sci. USA, 85:6949-6953 (1988); S.Rosenberg et al., New England J. Med., 323:570-578 (1990); K. Culver etal., Proc. Natl. Acad. Sci. USA, 88:3155-3159 (1991); G. Y. Wu and C. H.Wu, J. Biol. Chem., 263, No. 29:14621-14624 (1988); Wagner et al., Proc.Natl. Acad. Sci. USA, 87:3410-3414 (1990); Curiel et al., Human GeneTher., 3:147-154 (1992); Litzinger, Biochimica et Biophysica Acta,1104:179-187 (1992); Trubetskoy et al., Biochemica et Biophysica Acta,1131:311-313 (1992)). The present approach, within the context of a genetargeting mechanism either directed toward dividing tumor cells or tumorneovascularization, offers an improved means by which a small subset oftumor cells could be established within a growing tumor mass, whichwould mediate rapid tumor involution and necrosis after the appropriatesignal, i.e., after administration of the substrate (prodrug) for asuitable purine analog nucleoside cleavage enzyme, such as E. coli PNPpresent in or adsorbed to tumor cells.

Methods of Treatment

The method of treatment basically consists of providing to cells the PNPgene and then exposing the cells with the PNP gene or protein to anappropriate substrate which is converted to a toxic substance whichkills the cells expressing the PNP gene as well as those in the vicinityof the PNP gene expressing cells. The PNP gene can be administereddirectly to the targeted cells or systemically in combination with atargeting means, such as through the selection of a particular viralvector or delivery formulation. Cells can be treated in vivo, within thepatient to be treated, or treated in vitro, then injected into thepatient. Following introduction of the PNP gene into cells in thepatient, the prodrug is administered, systemically or locally, in aneffective amount to be converted by the PNP into a sufficient amount oftoxic substance to kill the targeted cells.

Treatment of Tumors

The E. coli PNP gene can also be used as part of a strategy to treatmetastatic solid tumors, such as melanoma, pancreatic, liver or coloniccarcinoma. No effective therapy for metastatic tumors of these typescurrently exists. In this method, plasmid DNA containing a PNP geneunder the control of tumor specific promoters is used. For example, thetyrosinase promoter is highly specific for mediating expression inmelanoma cells, and will not lead to gene expression in most tissuetypes. The PNP gene under the regulatory control of this promoter,therefore, should be activated predominantly within a melanoma tumor andnot elsewhere within a patient (see Example 11 and FIGS. 2A-D below).Promoters specific for other tumor types, for example, promoters activein the rapidly dividing endothelial cells present in all solid tumorscan be used to specifically activate PNP only within a primary ormetastatic tumor. In this method, plasmid DNA containing PNP under thecontrol of a tumor specific promoter is delivered to cells usingcationic liposomes. For example, based on animal studies, 100-400 mgplasmid DNA complexed to 1200-3600 micromoles of a 1:1 mixture of thelipids DOTMA (1,2-dioleyloxypropyhl-3-trimethyl ammonium bromide) andDOPE (dioleoyl phosphatidylethanolamine) could be used to deliver thePNP gene to tumor metastases in patients. A prodrug in the abovedescribed amounts can then be administered.

The PNP gene can be used to activate prodrugs in the treatment of humanbrain cancer. In this method, a cell line producing retroviralparticles, in which the viral particles contain the E. coli PNP gene, isinjected into a central nervous system (CNS) tumor within a patient. AnMRI scanner is used to appropriately inject the retroviral producer cellline to within the tumor mass. Because the retrovirus is fully activeonly within dividing cells and most of the dividing cells within thecranium of a cancer patient are within the tumor, the retrovirus isprimarily active in the tumor itself, rather than in non-malignant cellswithin the brain. Clinical features of the patient including tumor sizeand localization, determine the amount of producer cells to be injected.For example, a volume of producer cells in the range of 30 injections of100 microliters each (total volume 3 ml with approximately 1×10⁸producer cells/ml injected) are given under stereotactic guidance forsurgically inaccessible tumors. For tumors which can be approachedintraoperatively, 100 μl aliquots are again injected (at about 1×10⁸cells/ml) with total injected volumes up to 10 ml using E. coli PNP genetransfer, followed by MeP-dR administration. This strategy is designedto permit both bystander killing and toxicity to non-dividing cells andis thus designed for much greater tumor involution than previousattempts using HSV dThd kinase and ganciclovir.

The destruction of selected populations of cells can be achieved bytargeting the delivery of the bacterial PNP gene or other gene encodingan enzyme capable of cleaving purine analog from a purine analognucleoside (such as adenine from adenine-containing nucleosides asdescribed above). The natural tropism or physiology of viral vectors canalso be exploited as a means of targeting specific cell types. Forexample, retroviruses are well known to become fully active only inreplicating cells. This fact has been used as the basis for selectiveretroviral-mediated gene transfer to replicating cancer cells growingwithin a site where the normal (nonmalignant) cells are not replicatingin both animal and human clinical studies. Alternatively, the viralvector can be directly administered to a specific site such as a solidtumor, where the vast majority of the gene transfer will occur relativeto the surrounding tissues. This concept of selective delivery has beendemonstrated in the delivery of genes to tumors in mice by adenovirusvectors. Molecular conjugates can be developed so that the receptorbinding ligand will bind only to selective cell types, as has beendemonstrated for the lectin-mediated targeting of lung cancer.

Recently, it Was shown that intravenous injection of liposomes carryingDNA can mediate targeted expression of genes in certain cell types.Targeting of a gene encoding a purine analog nucleoside cleavage enzymeor expression of the gene to a small fraction of the cells in a tumormass followed by substrate administration could be adequate to mediateinvolution. Through the substantial bystander effect and killing ofnondividing cells demonstrated in the Examples, the present method canbe used to destroy the tumor.

Treatment of Virally Infected Cells

In addition to killing tumor cells, the methods described herein canalso be used to kill virally infected cells. In a virus-killingembodiment, the selected gene transfer method is chosen for its abilityto target the expression of the cleavage enzyme in virally infectedcells. For example, virally infected cells may utilize special viralgene sequences to regulate and permit gene expression, that is, virusspecific promoters. Such sequences are not present in uninfected cells.If the PNP gene is oriented appropriately with regard to such a viralpromoter, the cleavage enzyme would only be expressed within virallyinfected cells, and not other, uninfected, cells. In this case, virallyinfected cells would be much more susceptible to the administration ofMeP-dR or other substrates designed to be converted to toxic form bynon-human or modified human purine nucleoside cleavage enzyme.

Administration of Genetically Engineered Cells

For certain applications, cells that receive the PNP gene are selectedand administered to a patient. This method most commonly involves exvivo co-transfer of both the gene encoding the cleavage enzyme, such asthe bacterial PNP gene, and a second gene encoding a therapeutic proteingene. The cells that receive both genes are reinfused into the hostpatient where they can produce the therapeutic protein until theprodrug, such as MeP-dR, is administered to eliminate the engineeredcells. This method should be useful in “cell therapies”, such as thoseused on non-replicating myoblasts engineered for the production oftyrosine hydroxylase within the brain (Jiao et al., Nature, 362:450(1993)).

Direct Delivery of the PNP Enzyme to Cells

The bystander killing conferred by the bacterial PNP protein plusprodrug combination can also be achieved by delivering the PNP proteinto the target cells, rather than the PNP gene. For example, a PNP enzymecapable of cleaving purine analog nucleosides as described above, ismanufactured by available recombinant protein techniques usingcommercially available reagents. As one example of a method forproducing the bacterial PNP protein, the E. coli PNP coding sequence isligated into the multiple cloning site of pGEX-4T-1 (Pharmacia,Piscataway N.J.) so as to be “in frame”, with theglutathione-s-transferase (GST) fusion protein using standard techniques(note that the cloning site of this vector allows insertion of codingsequences in all three possible translational reading frames tofacilitate this step). The resulting plasmid contains the GST-PNP fusioncoding sequence under transcriptional control of the IPTG-inducibleprokaryotic tac promoter. E. coli cells are transformed with therecombinant plasmid and the tac promoter induced with IPTG. IPTG-inducedcells are lysed, and the GST-PNP fusion protein purified by affinitychromatography on a glutathione Sepharose 4B column. The GST-PNP fusionprotein is eluted, and the GST portion of the molecule removed bythrombin cleavage. All of these techniques and reagents are provided ina commercially available kit (Pharmacia, Piscataway, N.J., catalog no.27-457001). Other methods for recombinant protein production aredescribed in detail in published laboratory manuals. Since the bacterialPNP activates the prodrugs into diffusible toxins, it is only necessaryto deliver the PNP protein to the exterior of the target cells prior toprodrug administration. The PNP protein can be delivered to targets by awide variety of techniques. One example would be the direct applicationof the protein with or without a carrier to a target tissue by directapplication, as might be done by directly injecting a tumor mass withinan accessible site. Another example would be the attachment of the PNPprotein to a monoclonal antibody that recognizes an antigen on the tumorsite. Methods for attaching functional proteins to monoclonal antibodieshave been previously described. The PNP conjugated monoclonal antibodyis systemically administered, for example, intravenously (IV), andattaches specifically to the target tissue. Subsequent systemicadministration of the prodrug will result in the local production ofdiffusible toxin in the vicinity of the tumor site. A number of studieshave demonstrated the use of this technology to target specific proteinsto tumor tissue. Other ligands, in addition to monoclonal antibodies,can be selected for their specificity for a target cell and testedaccording to the methods taught herein.

Another example of protein delivery to specific targets is that achievedwith liposomes. Methods for producing liposomes are described e.g.,Liposomes: A Practical Approach). Liposomes can be targeted to specificsites by the inclusion of specific ligands or antibodies in theirexterior surface, in which specific liver cell populations were targetedby the inclusion of asialofetuin in the liposomal surface (Van Berkel etal., Targeted Diagnosis and Therapy, 5:225-249 (1991)). Specificliposomal formulations can also achieve targeted delivery, as bestexemplified by the so-called StealthJ liposomes that preferentiallydeliver drugs to implanted tumors (Allen, Liposomes in the Therapy ofInfectious Diseases and Cancer, 405-415 (1989)). After the liposomeshave been injected or implanted, unbound liposome is allowed to becleared from the blood, and the patient is treated with the purineanalog nucleoside prodrug, such as MeP-dR, which is cleaved to MeP bythe E. coli PNP or other suitable cleavage enzyme at the targeted site.Again, this procedure requires only the availability of an appropriatetargeting vehicle. In a broader sense, the strategy of targeting can beextended to specific delivery of the prodrug following either PNPprotein, or gene delivery.

Administration of Substrates

The formula of Freireich et al., Cancer Chemother. Rep., 50:219-244,(1966) can be used to determine the maximum tolerated dose of substratefor a human subject. For example, based on systemically administereddose response data in mice showing that a dose of 25 mg (Mep-dR) per kgper day for 9 days (9 doses total) resulted in some toxicity but nolethality, a human dosage of 75 mg MeP-dR/m² was determined according tothe formula: 25 mg/kg×3=75 mg/m². This amount or slightly less shouldresult in maximal effectiveness of tumor cell killing in humans withoutkilling the subject. This standard of effectiveness is accepted in thefield of cancer therapy. However, more preferred is a range of fromabout 10% to 1% of the maximum tolerated dosage (for example, 7.5mg/m²-0.75 mg/m²). Furthermore, it is understood that modes ofadministration that permit the substrate to remain localized at or nearthe site of the tumor will be effective at lower doses than systemicallyadministered substrates.

The substrate may be administered orally, parenterally (for example,intravenously), by intramuscular injection, by intraperitonealinjection, or transdermally. The exact amount of substrate required willvary from subject to subject, depending on the age, weight and generalcondition of the subject, the severity of the disease that is beingtreated, the location and size of the tumor, the particular compoundused, its mode of administration, and the like. An appropriate amountmay be determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein. Generally, dosage willpreferably be in the range of about 0.5-50 mg/m², when consideringMeP-dR for example, or a functional equivalent.

Depending on the intended mode of administration, the substrate can bein pharmaceutical compositions in the form of solid, semi-solid orliquid dosage forms, such as, for example, tablets, suppositories,pills, capsules, powders, liquids, or suspensions, preferably in unitdosage form suitable for single administration of a precise dosage. Thecompositions will include an effective amount of the selected substratein combination with a pharmaceutically acceptable carrier and, inaddition, may include other medicinal agents, pharmaceutical agents,carriers, or diluents. By “pharmaceutically acceptable” is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected substrate withoutcausing significant undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucroseand magnesium carbonate. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving or dispersingan active compound with optimal pharmaceutical adjuvants in anexcipient, such as water, saline, aqueous dextrose, glycerol, orethanol, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, for example, sodium acetate ortriethanolamine oleate. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences.

For oral administration, fine powders or granules may contain diluting,dispersing, and/or surface active agents, and may be presented in wateror in a syrup, in capsules or sachets in the dry state or in anonaqueous solution or suspension wherein suspending agents may beincluded, in tablets wherein binders and lubricants may be included, orin a suspension in water or a syrup. Where desirable or necessary,flavoring, preserving, suspending, thickening, or emulsifying agents maybe included. Tablets and granules are preferred oral administrationforms, and these may be coated.

Parenteral administration is generally by injection. Injectables can beprepared in conventional forms, either liquid solutions or suspensions,solid forms suitable for solution or prior to injection, or assuspension in liquid prior to injection or as emulsions.

Cells Expressing E. coli PNP Substrate

The effect of MeP-dR on human colon carcinoma cells expressing E. coliPNP substrate was demonstrated. MeP-dR was chosen because it is 20-foldless toxic than 6-methylpurine (MeP) to HEp-2 cells and it has been usedto detect cultures infected with mycoplasma, because mycoplasma expressan enzyme similar in function to E. coli PNP.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Cell Lines

T-84 colon carcinoma cells were grown in Dulbecco's modified Eaglemedium containing F12 nutrient medium (DMEM/F12) (GIBCO/BRL,Gaithersburg, Md.) in 6 well trays to a density of approximately 1-2×10³cells/well (−20% confluency).

EXAMPLE 2

Toxicity of MeP and MeP-dR within Colon Carcinoma Cells

Untransfected T-84 colon carcinoma cells were treated with increasingconcentrations of either MeP-dR or MeP. After 5 days the cells wereremoved from each well and the number of dye excluding cells weredetermined with the aid of a hemacytometer. Cells were studied both atpassage 48 (p. 48) and passage 61 (p. 61). MeP was obtained from SigmaChemical Company (St. Louis, Mo.). MeP-dR was synthesized by standardmethods as described (J. A. Montgomery and K. Howson, J. Med. Chem.,11:48-52 (1968)). The nucleoside and base were dissolved in serum freeDMEM/F12 at a concentration of 1 mg/ml and added directly to 1 mlDMEM/F12 with 10% fetal bovine serum at the concentrations describedbelow in order to cover 1-2×10⁵ cells/well.

Initial cytopathic effects due to MeP were observed within 24 hours (forexample, rounding of cells, with some cells detaching from plate).Viable cells were counted 5 days following addition of drug. The higherconcentrations (3.75 μM-75 μM) of MeP resulted in cell lysis andcomplete loss of cellular architecture, leaving only cellular debriswithin wells by day 2 following treatment. Trypan blue exclusion wasused to confirm viability in cells retaining recognizable structure atall concentrations studied. At lower concentrations MeP-dR did not causeany appreciable cell death and higher concentrations (200 and 400 μM)less than half of the cells were killed. If the toxicity of MeP-dR isdue to very low levels of liberation of MeP by human PNP, thencombination with selective inhibitors of human PNP could prevent thistoxicity.

The relative toxicity of the prodrug, MeP-dR, and the product, MeP, onwild type melanoma cell viability, was tested. Mel-1 cells wereincubated in various concentrations of MeP-dR and MeP for five days. TheMel-1 cells were unaffected by concentrations of MeP-dR as high as 50μg/ml while concentrations of the MeP as low as 0.5 μg/ml were nearly100% lethal. Similar results have been obtained in T-84, B16, and 16/Ccells. Both MeP-dR and MeP are stable under tissue culture conditions asmeasured by HPLC analysis of supernatants.

EXAMPLE 3

Synthesis of E. coli PNP Expression Vectors

A bacterial PNP-encoding sequence was inserted into a plasmid expressionvector. E. coli (strain, JM101) chromosomal DNA template was obtainedusing the method described in N. J. Gay, J. Bacteriol., 158:820-825(1984). Two PCR primers GATCGCGGCCGCATGGCTACCCCACACATTAATGCAG (SEQ IDNO: 1) and GTACGCGGCCGCTTACTCTTTATCGCCCAGCAGAACGGA-TTCCAG (SEQ ID NO: 2)were used to define the full length coding sequence of the E. coli DeoDgene and to incorporate NotI sites at both 50 and 30 ends of the desiredproduct. After 30 cycles of amplification (94° C.×1 minute denaturation,50° C.×2 minute annealing, and 72° C.×3 minute elongation using 1 ngtemplate, 100 μl of each primer in a 100 μl reaction mixture containing2.5 units taq polymerase, 200 μM each dNTP, 50 mM KCl, 10 mM Tris Cl (pH8.3), 1.5 mM MgCl₂ and 0.01% gelatin (weight/vol)), a single PCR productof the predicted size (716 base pairs) was obtained. This product wasextracted with phenol/chloroform, precipitated with ethanol, digestedwith NotI, and gel purified using the Gene clean kit (Bio. 101, LaJolla, Calif.).

The amplified bacterial PNP sequence was added to a plasmid eukaryoticexpression vector. In order to obtain a vector capable of directingeukaryotic expression of E. coli PNP, the LacZ gene was excised frompSVB (Clontech, Palo Alto, Calif.) by digestion with NotI, the vectorbackbone was dephosphorylated (calf intestinal alkaline phosphatase,GIBCO BRL, Gaithersburg, Md.) and gel purified as above. The PNP insert,prepared as above, was then ligated into the Notl ends of the plasmidbackbone in order to create a new construct with PNP expressioncontrolled by the SV-40 early promoter. Correct recombinants (andorientation of inserts) were confirmed by restriction mapping (usingtwelve restriction digests which cut in both vector and insert), and byreamplification of the full length insert from recombinant plasmid usingthe primers described above. This procedure yielded the plasmid SV-PNP.

EXAMPLE 4

Transfection of T-84 Colon Carcinoma Cells

Cationic liposome mediated gene transfer was used to transfect T-84colon carcinoma cells. Briefly, 6 μg of plasmid containing PNP or LacZwas added to 10 μg of a 1:1 molar mixture of DOTMA/DOPE (LipofectinJ(GIBCO/BRL, Gaithersburg, Md.)) in a final volume of 200 μl DMEM/FI2serum free medium. After a 10 minute incubation at room temperature, theDNA-lipid mixture was added to 500 μl serum free medium and was used tocover the cells within a tray. Four hours later, transfection medium wasremoved from each well and 2 ml DMEM/FI2 with 10% fetal bovine serum wasadded.

EXAMPLE 5

Transfection Efficiency

The LacZ gene was transfected into T-84 cells as described above.Briefly, using a lipid-mediated gene transfer protocol identical to thatdescribed above, 6 μg of plasmid containing the E. coli LacZ gene underthe control of the SV-40 early promoter was transferred into 1-2×10⁵T-84 cells. 48 hours after transfection, cells were washed 3 times inPBS, fixed at 4° C.×10 minutes in 0.2% glutaraldehyde, (in 80 mMNaHPO₂), rinsed 2 times with PBS, and then stained in a solutioncontaining 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 1.3 mM MgCl₂, 3 mM K₃Fe(CN)₆, 3mM K₄Fe(CN)₆ and 1 mg/ml x-gal(5-bromo-4-chloro-3-indolyl-β-D-galactosidase). 12 hours after staining,0.1-1% of the cells treated with β-galactosidase DNA stained positivefor gene expression.

X-gal staining of these cells two days after transfection indicated anoverall transfection efficiency of 0.1-1% (as determined by percentageof blue cells). No positive cells were observed in untreated T-84 cells,in cells treated with lipid alone, or plasmid DNA alone. Similarconclusions were reached using a LacZ reporter gene containing a nucleartargeting sequence and leading to nuclear staining of recombinant cells.

EXAMPLE 6

Toxicity of MeP-dR-mediated by E. coli PNP Expression Vectors

Forty-eight hours following transfection, fresh medium was added andMeP-dR (1 mg/ml in PBS) was added directly to the cells to achieve thedesired final concentrations. Cell viability was measured 5 daysfollowing treatment as described for the MeP-dR toxicity study.

In one experiment, MeP-DR (160 μM) was added to wells containinguntransfected cells, or cells transfected with 10, 20, or 40 μg (FIG. 1)of cDNA containing either the E. coli PNP or LacZ genes under control ofthe SV-40 early promoter (in otherwise comparable vector contexts).After 5 days the cells were removed from each well and the number of dyeexcluding cells were determined with the aid of a hemacytometer. 30-50%toxicity due to the DOTMA-DOPE transfection protocol is acceptable forcationic liposome mediated gene transfer to T-84 in vitro when performedunder optimal conditions. The results of this study are shown in FIG. 1.

In an additional experiment, approximately 2×10⁵ cells per well weretransfected as above using 6 μg of plasmid containing E. coli PNP cDNA.Two days after transfection, varying concentrations of MeP-dR (0, 2, 4,20, 40 and 160 μM) were added to the wells, and after 5 days the dyeexcluding cells were counted with the aid of a hemacytometer.Concentrations of MeP-dR as low as 4 μM resulted in greater than 80%inhibition of cell growth.

An experiment was also performed in triplicate in which 2×10⁵ cells perwell were transfected with LacZ or PNP using the protocol described fortransfection. Two days after transfection, 16 μM MeP-dR was added to oneset of the cultures transfected with PNP and one set of the culturestransfected with LacZ. The other PNP and LacZ transfected cultures didnot receive drug. The results demonstrate minimal cell killing in allcultures except the PNP transfected, MeP-dR treated culture.

In the above experiments, MeP-dR (160 μM) was minimally toxic to thecells that were not transfected. While expression of the LacZ gene hadno influence on toxicity mediated by MeP-dR, MeP-dR killed virtually allof the cells transfected with the E. coli PNP (FIG. 1). Substantialkilling could also be seen with 16 μM MeP-dR after PNP transfection.These results indicate that low efficiency expression of E. coli PNPcDNA (expression in less than 1% of tumor cells) was adequate for nearly100% transfected cell and bystander cell killing. In addition, becausediffusion of MeP into the medium covering the cells could have asubstantial dilutional affect, it may be that an even lower fraction oftumor cells expressing E. coli PNP in vivo might be able to mediatetumor cell necrosis in the presence of MeP-dR.

EXAMPLE 7

Activity of E. coli PNP on MeP-dR in Cell Extracts

The toxicity of MeP-dR in T-84 cells expressing the E. coli PNP activitywas measured in transfected T-84 cells. Briefly, T-84 cells transfectedwith 6 μg of plasmid containing either the E. coli PNP gene or the LacZ(β-galactosidase) gene as described above were collected bycentrifugation 48 hours after transfection and resuspended in 3 volumesof 0.01 M potassium phosphate (pH 7.4), followed by incubation on icefor 15 minutes. The pellet was homogenized, and the sample wascentrifuged at 100,000×g for 60 minutes. PNP activity was measured in100 μl volumes containing 50 mM potassium phosphate (pH. 7.4), 100 μM ofMeP-dR, and 1 mg/ml of protein from the cell extract. After incubationfor 24 hours at 25° C., the reaction was stopped by boiling, theprecipitated proteins were removed by centrifugation, and the reactionmixture was subjected to HPLC by injection onto a Spherisorb ODSI (5 μm)column (Keystone Scientific Inc., State College, Pa.). The MeP-dR andMeP were eluted with a 30 min isocratic gradient of 50 mM ammoniumdihydrogen phosphate buffer (pH 4.5)/acetonitrile (95/5; v/v) at a flowrate of 1 ml/minute. MeP-dR and MeP were detected by their absorbance at254 nm.

Approximately 24% of the MeP-dR was converted to MeP in extracts fromthe T-84 colon carcinoma cells transfected with the E. coli PNP gene,whereas no conversion occurred in cell extracts from colon carcinomacells transfected with the LacZ gene. Total PNP activity (human+E. coli)measured using inosine as substrate was not changed in T-84 cellstransfected with E. coli PNP. Thus, despite the relatively low level ofexpression of the E. coli PNP in the transfected cells, a sufficientamount of the MeP-dR was converted to kill all of the cells.

EXAMPLE 8

Detection of MeP in Medium of T-84 Cells Transfected With E. coli PNP

MeP-dR (160 μM) was added 48 hours after transfection of T-84 cells withthe E. coli PNP gene. Five days after the addition of MeP-dR, the mediumwas collected, and the proteins were precipitated by boiling. Aftercentrifugation, the medium was analyzed for the appearance of MeP byreverse phase HPLC as described above.

MeP was detected only in the culture medium of T-84 cells transfectedwith E. coli PNP. More than 75% of the MeP-dR was converted to MeP overa 5 day period in E. coli PNP transfected cells, but not in LacZtransfected cells. These results have significance, because theyindicated that 1) untransfected and mock transfected colonic carcinomacells lack an enzymatic mechanism for conversion of MeP-dR to MeP, 2) aspredicted, MeP was readily released into the extracellular medium, so asto establish effective bystander killing, and 3) the extracellularconcentrations of MeP generated by recombinant PNP were sufficient tofully explain the bystander killing which was observed. In addition,these results establish that SV-40 driven expression of the prokaryoticPNP in eukaryotic cells (as with the E. coli LacZ) leads to a highlyactive and functional enzyme. Because E. coli PNP is believed toassemble as a homohexamer in prokaryotic cells, the mechanisms of E.coli PNP oligomerization are likely to be compatible with eukaryoticprotein synthesis.

EXAMPLE 9

Toxicity to Nondividing Cells

Results from experiments indicate that MeP is able to killnon-proliferating cells. This distinguishes MeP from most otherantitumor agents. In the first experiment, CEM cells were cultured in 1%serum instead of the normal 10% serum for 48 hours. Under theseconditions, the cells stop growing and the cell numbers stabilize at 1.5to 2 times the original cell numbers. Cell growth continues when cellsare returned to culture medium containing 10% serum. Addition of MeP ata final concentration of 10 μg/ml to CEM cell cultures after 48 hours ofincubation with 1% serum caused a decline in cell numbers toapproximately 25% of their original number which indicated that MeP wastoxic to non-proliferating cells.

In the second experiment, the effect of MeP on the incorporation ofthymidine into DNA, uridine into RNA, and leucine into protein wasdetermined. RNA and protein synthesis were affected most by treatmentwith MeP. Effects on DNA synthesis occurred only after effects on RNAand protein synthesis were evident. These results indicated that theinhibitory effect of MeP on either RNA or protein synthesis wasresponsible for its toxicity. These two functions are vital to all cellsregardless of their proliferative state, which indicates that MeP shouldbe toxic to both proliferating and non-proliferating cells. Resultsconfirming these conclusions were also obtained in MRC-5 which are anon-transformed human diploid fibroblast cell line derived fromembryonic lung cells.

EXAMPLE 10

Additional Useful Recombinant Vectors

A recombinant retrovirus was made by adding the bacterial PNP sequencesto a plasmid retroviral transfer vector that was subsequently passedthrough packaging cell lines for the production of virus. The retroviralvector, pLNSX (Miller and Rosman, Bio Techniques, 7:980-991 (1989)),contains a cloning site that is just 30 to an SV40 early promoter whichwill direct transcription of a coding sequence inserted within thecloning site. The bacterial PNP sequence was ligated into linearizedpLNSX. The ligation mixture was used to generate bacterial transformantsthat were identified by colony DNA analysis, and one clone (pLN/PNP)containing the PNP coding sequence in a 50 to 30 orientation relative tothe SV40 promoter was amplified by standard techniques and purified withcesium chloride gradient centrifugation. The plasmid was transfected bylipid-mediated gene transfer into the ψ2 packaging cell line. Thesupernatant from these cells was harvested 48 hours later, clarified by0.45 μM filtration and applied to additional ψ2 packaging cell line. In24-36 hours, the cells were enzymatically detached and plated at adensity ⅕ the original density in media supplemented with G418 (1 g/L).Virus producing cells appeared as colonies 7-10 days later that wereisolated with cloning rings and assessed for quantity and fidelity ofrecombinant virus production.

A recombinant adenovirus was made by adding the bacterial PNP sequencesto a plasmid adenoviral vector that was subsequently passed through acell line (293) for the production of virus. The adenoviral plasmidvector, pACCMV (Kolls et al., Proc. Natl. Acad. Sci. USA, 91:215-219(1993)) was linearized with EcoRI and HindIII at the multiple cloningsite which is operably linked to the cytomegalovirus (CMV) immediateearly promoter. The bacterial PNP-encoding sequence was excised from theSV/PNP plasmid using Notl, and the fragment gel purified and ligatedinto the NotI site of pSL1180 (Pharmacia, Piscataway, N.J.) to producethe plasmid designated pSL/PNP. The PNP encoding sequence was excisedfrom pSL/PNP with EcoRI and HindIII, gel purified, and ligated into theEcoRI and HindIII site of pACCMV to make the new plasmid designatedpACCMV/PNP. Transfection of the pACCMV/PNP into cells conferreddose-dependent toxicity following exposure to the MeP-dR prodrug,confirming that the CMV promoter directed the production of therapeuticlevels of the bacterial PNP. The pACCMV/PNP was cotransduced with thepJMI7 vector into human embryonal carcinoma 293 cells that containadenoviral E1A sequences necessary for viral replication.

EXAMPLE 11

Tyrosinase Promoter Sequence-directed Expression Plasmids

The human tyrosinase regulatory sequence was amplified by the polymerasechain reaction (PCR) from human genomic DNA. The genomic DNA wasobtained from nucleated human blood cells by standard techniques. PCRprimers A (GAT CGC TAG CGG GCT CTG AAG ACA ATC TCT CTC TGC (SEQ IDNO.3)) and B (GAT CGC TAG CTC TTC CTC TAG TCC TCA CAA GGT CT (SEQ IDNO.4)) amplified bp −451, to +78 with the addition of NheI restrictionenzyme sites at each end using the sequence of Kikuchio et al., Biochim.Biophys. Acta. 2009:283-286 (1989). The PCR reaction used the followingconditions for 30 cycles: 94° C.×1 min, 50° C.×2 min, 72° C.×3 minutes.The final product was clarified by phenol/chloroform extraction,digested with NheI, gel purified, and ligated into the NheI cloning siteof the commercial luciferase vector, pGL2 Basic (Promega, Madison, Wis.)by standard techniques. Recombinants were screened by restrictionmapping and a correctly oriented clone was identified (Tyr-Luc). Aplasmid with the tyrosinase promoter in reverse orientation(Rev-Tyr-Luc), for use as a negative control, was also selected. Acontrol vector (SV-LUC) containing the SV-40 virus early promoter andSV-40 enhancer region driving the expression of firefly luciferase (pGL2control vector, Promega, Madison, Wis.) was used to verify successfultransfection of cells. To create a plasmid in which the tyrosinasepromoter controlled PNP expression, the PNP gene was substituted forluciferase in the Tyr-Luc. This was accomplished using a XhoI/Sal1digest to excise the full length PNP gene from SV-PNP, followed byinsertion of this fragment into the XhoI/Sal1 sites remaining after aXhoI/Sal1 digest to remove the luciferase gene from Tyr-Luc. Thetyrosinase reporter constructs were tested in transient assays. TheLipofectinJ (GIBCO/BRL, Gaithersburg, Md.) transfection protocol wasused for all luciferase reporter gene experiments. Cells were seeded at50% confluency in six-well plates and allowed to grow overnight.Immediately prior to transfection each well was washed three times withsterile phosphate buffered saline (PBS). A single well of a six-wellplate was transfected with a ratio of 10 μg liposomes/10-20 μg ofplasmid DNA, depending on the cell line. Liposome/DNA complexes wereprepared according to manufacturer's instructions. The liposome/DNAcomplexes were mixed with serum free media (SFM) and a total volume of700 μl was placed in a single well of a six-well plate. After incubationat 37° C. for 14 to 16 hours, the transfection mixture was aspirated and2 ml of complete media was added. The cells were harvested after 48additional hours and luciferase activity was determined using theinstructions and reagents of a commercial kit (Luciferase Assay System,Promega, Madison, Wis.). Luciferase reporter gene expression wasassessed 48, hours following transfection of various carcinoma celllines (melanoma, liver, colon, prostate, myeloma, glial, HeLa) with aconstruct containing a promoterless luciferase vector (“Basic”); aluciferase gene linked to a human tyrosinase promoter in reverseorientation (incorrect orientation to transcribe the luciferase gene)(Rev-Tyr-Luc); a luciferase gene operably linked to the constitutiveSV40 early promoter (SV-Luc); or a luciferase gene operably linked to ahuman tyrosinase promoter (correct orientation to transcribe theluciferase gene) (Tyr-Luc).

As shown in FIGS. 2A-D, the tyrosinase transcriptional promoter sequencespecifically restricted expression of the luciferase reporter gene towhich it was operably linked, to melanoma cells (Mel-1 and Mel-21). Incontrast, the SV40 early promoter constitutively expressed theluciferase gene to which it was operably linked in all transfectedcarcinoma cell lines. The results demonstrate that tissue-specificpromoter sequences can be used to transcriptionally target theexpression of a heterologous enzyme to a specific tumor.

Luciferase activity in Mel-1 and Mel-21 cells transfected with theTyr-Luc construct was comparable to luciferase activity generated bytransfection with a plasmid utilizing the SV-40 early promoter tocontrol luciferase gene expression (SV-Luc) (FIG. 2, Panel A). Bothnegative controls (luciferase without promoter (Basic) and luciferasewith tyrosinase promoter sequences inserted in the reverse orientation(Rev-Tyr-Luc)) gave negative results. Negligible Tyr-Luc activity wasseen in five additional human cell lines (T-84-colon cancer, U373-glial,HeLa-cervical carcinoma, RPMI 8226-myeloma, GP6FS-prostate), which allshowed substantial SV-40 driven reporter gene activity (FIG. 2, PanelB-D). In a sixth cell line, Hep G2 (derived from human liver), theSV-Luc was 28 fold more active than the Tyr-Luc. However, the Tyr-Lucvector had activity above background in the Hep G2 cells. Because thepromoterless luciferase vector resulted in similar luciferase activity,luciferase activity in Hep G2 cells is likely to be nonspecific and dueto cryptic promoters or enhancers present within the vector itself,rather than nonspecific regulation by the human tyrosinase promoter.

To eliminate possible toxicity associated with the non-hydrolyzablecationic lipid component of the LipofectinJ, an alternative liposometransfection vehicle was used in the killing experiments. A liposomevehicle consisting of a 1:1 (weight/weight) mixture of the cationiclipid DOTAP (1,2-dioleoyloxy-3-(trimethylammonium)-propane) and theneutral lipid DOPE (dioleoyl-phosphatidylethanolamine) (Avanti PolarLipids) display transfection properties similar to LipofectinJ, but withless toxicity (data not shown). DOTAP/DOPE liposomes were prepared bymixing 0.5 mg of DOTAP and 0.5 mg of DOPE and evaporating the chloroformsolvent. Following the addition of 500 μl of cyclohexane, the mixturewas placed on dry ice and lyophilized. One ml of sterile water was addedto the powdered lipids and the solution was vortexed every 5 minutes for30 minutes. T-84 or Mel-1 cells were seeded at 30% confluency in 24-wellplates and allowed to grow overnight. Immediately prior to transfection,each well was washed three times with sterile PBS. To transfect a singlewell of a 24-well plate, 7.5 μg of DOTAP/DOPE (1 μg/μl) was mixed with1.875 μg of plasmid DNA (1 μg/μl) and incubated for 15 minutes.Following a 15 minute incubation, the liposome/DNA complexes were mixedwith 266 μl of SFM and added to a single well of a 24-well plate. Theplates were incubated for four hours at 37° C., and then thetransfection mixture was aspirated and replaced with 500 μl of completemedia. Using this protocol, no significant toxicity due to transfectionwas observed.

In cells that received the PNP or control plasmids, the media waschanged two days after transfection and MeP-dR(6-methylpurine-deoxyriboside) added to the appropriate wells to a finalconcentration of 30 μg/ml. Four days later, the cells were fed by addingfresh media with MeP-dR (30 μg/ml) to the wells without removing the oldmedia. Two days later (day 6), the cells were washed once with PBS,resuspended, and counted in a 20% solution of trypan blue reagent(Trypan Blue Stain 0.4%, Gibco-BRL, Gaithersburg, Md.) using ahemacytometer.

Both T-84 colon carcinoma cells and Mel-1 melanoma cells weretransfected using DOTAP/DOPE liposomes (FIG. 3) with the SV-PNPconstruct, in which the constitutive SV40 early promoter is operablylinked to the bacterial PNP gene; or the Tyr-PNP, in which the melanomaspecific tyrosinase promoter is operably linked to the bacterial PNPgene; or the Tyr-Luc (see above); or not transfected with anyrecombinant construct (“no txf”). Only melanoma cells (Mel-1)transfected with the Tyr-PNP construct were susceptible to killing uponadministration of the prodrug MeP-dR purine analog nucleoside asdemonstrated by comparing FIG. 3A, transfected T-84 colon carcinomacells, with FIG. 3B, transfected Mel-1 melanoma cells. In contrast, whenthe constitutive SV40 early promoter was operably linked to thebacterial PNP gene (SV-PNP construct), both T-84 colon carcinoma andMel-l melanoma cells transfected with the SV-PNP construct weresusceptible to killing upon administration of the prodrug MeP-dR. Theseresults demonstrate that transcriptional targeting of the expression ofa purine analog nucleoside cleavage gene permits selective killing ofspecific tumor cells. Cell death under these conditions correlates withthe amount of MeP generated by the action of recombinant E. coli PNP onMeP-dR. The transfection of plasmid containing either a cytoplasmic or anuclear targeted β-galactosidase gene under the same conditionsindicated a low transfection efficiency (<0.1% of cells positive forLacZ).

EXAMPLE 12

Method for Identifying Candidate Prodrugs for Bacterial PNP

The following method is useful to identify substrates (prodrugs) thatare cleaved more efficiently by the bacterial PNP than by mammalian PNP.Prodrugs identified by this method can then be further assessed byanimal studies for determination of toxicity, suitability foradministration with various pharmaceutical carriers, and otherpharmacological properties.

The method quantitatively measures the cleavage of substrates in vitro.The purine analog nucleosides (0.1 or 1.0 mM) were incubated in 500 μlof 100 mM HEPES, pH 7.4, 50 mM potassium phosphate, and with 100 μg/mlE. coli PNP or 0.1 unit/ml human PNP. The reaction mixtures wereincubated at 25° C. for 1 hour, and the reactions stopped by boilingeach sample for 2 minutes. The cleavage of [¹⁴C]inosine by each enzymewas determined as a positive control. Each sample was analyzed byreverse phase HPLC to measure conversion from substrate to product. Thenucleoside and purine analogs were eluted from a Spherisorb ODSI (5 μm)column (Keystone Scientific, Inc., State College, Pa.) with a solventcontaining 50 mM ammonium dihydrogen phosphate (95%) and products weredetected by their absorbance at 254 nm, and were identified by comparingtheir retention times and absorption spectra with authentic samples.

By this analysis, MeP-dR, 2-F-dAdo,1-deaza-2-amino-6-Cl-purine-riboside, 2-F-50-deoxyadenosine,2-Cl-20-deoxyadenosine were all shown to be good substrates forbacterial PNP and poor substrates for the mammalian PNP, and thus arepreferred candidate prodrugs which are eligible for further assessmentfor use in the methods and compositions described herein to treatmalignancies (MeP-dR is a suitable prodrug, as noted above). Substrates50-amino-50-deoxyadenosine, F-araA, and α-adenosine were moderatesubstrates for bacterial PNP and poor substrates for the mammalian PNP.Substrates xylosyl methylpurine, 2-Cl-20-F-20-deoxyadenosine,2-F-20-F-20-deoxyadenosine, and 7-ribosyl-6-mercaptopurine were poorsubstrates for both enzymes, and therefore would not be candidateprodrugs in conjunction with unmodified E. coli PNP. Similarly,substrates 7-ribosylhypoxanthine and thioguanosine were moderate to goodsubstrates for both enzymes and also would not be candidate prodrugs fortreating tumors using the compositions and methods described herein.

2-F-dAdo and F-araA have demonstrated antitumor activity not related tothe production of fluoroadenine. Therefore, in methods described herein,the antitumor activity of these two substrates is likely to bepotentiated by metabolism by the E. coli PNP. In addition, themetabolism and toxicity of these two agents can be prevented byincubation in the presence of 2′-deoxycytidine.

Thus, by combining these substrates with 2′-deoxycytidine, antitumoractivity related only to the production of fluoroadenine is possible.

TABLE II Screening of nucleotides as substrates for E. coil PNP Percentof substrate cleaved by: E. coli PNP Human PNP substrate 100 μM 1 mM 100μM 1 mM I. Nucleosides that are good substrates for E. coli PNP, but areat best poor substrates for human PNP. MeP-dR 93(87)29(24) 0 091(86)45(21) 0(86) 0(47) FdAdo 56(69)14(18)0(70) 0(30) 60(86)38(21)0(86) 0(47) 1-deaza-2-amino- 62(87)16(23) 0(88) 0(52) 6-Cl-purine-41(86)15(21) 0(86) 0(47) riboside 2-F-5′-deoxy- 81(86)30(21) 0(88) 0(50)adenosine 65(86)44(21) 0(86) 0(47) 2-Cl-2′-deoxy- 41(86)- 0(87) —adenosine 7-ribosyl-3- 88(91*) 67(43*) 0(0*) 0(0*) deazaguanine 84(90*)83(39*) 0(95*) 0(43**) #80(85**) — 0(87**) — 7-ribosyl-6- 0 0 0 0mercaptopurine**** 0(86) 0(21) 0(86**) 0(47) 500 μM #45(65**) 35(16**)0(87**) 0.37(40**) #10(85**) 0(87**) II. Nucleosides that are moderatesubstrates for E. coli PNP, but are at best poor substrates for humanPNP. 5′-amino-5′-deoxy- 5(86) 1(19) 0(89) 0(53) adenosine 9(86) 5(21)0(86) 0(47) #29(85**) — 0(87**) — F-araA 3(86) 3(21) 0(88) 0(50) 5(86)12(21) 0(86) 0(47) α-adeno- 0(86) 0(21) 0(88) 0(50) sine 3(86) 2(21)0(86) 0(47) #0(85**) — 0(87**) — III. Nucleosides that are at best poorsubstrates for both enzymes. xylosylmethyl- 0(86) 0(21) 0(88) 0(50)purine 0(86) 0(21) 0(86) 0(47) xylosyl adenine 0(78) — 0(81) — 1(56) —0(82) — 2-Cl-2′-F-2′-deoxy- 0(86) 0(21) 0(88) 0(50) adenosine 0(86)0(21) 0(86) 0(47) 2-F-2′-F-2′-deoxy- 0(86) 0(21) 0(88) 0(50) adenosine0(86) 0(21) 0(86) 0(47) 2′,3′-dideoxy 1.6 (64**) 0(15**) 1.2(85**)0.2(49**) adenosine*** #0(85**) — 0(87**) — 2′,3′-dideoxy- 2.7(64**)3(15**) 1.1(85**) 2.4(49**) inosine*** #0(85**) — 0(87**) — 3′-deoxy0(62**) 0(16**) 0(87**) 0(45**) adenosine #0(85**) — 0(87**) —5′-carboxamide #1.2(78) — 0(81) — of adenosine 0.1(56) — 0(82) —Isopropylidine #1(78) — 0(81) — of the 0(56) — 0(82) — 5′-carboxamide ofadenosine IV. Nucleosides that are substrates for both enzymes.7-ribosyl-hypo- 16(86)30(21)3(86) 5(47) xanthine 49(86)38(21)73(86)73(47) thioguanosine 49(86)38(21)73(86) 48(47)

In Table II, above, each of the numbers represent the percent conversionof the purine analog nucleoside by the phosphorylase indicated. Thenumbers in parentheses are percent conversion of the inosine tohypoxanthine in the same experiment. “*” indicates that MeP-dR was usedas the control agent in place of inosine. “**” indicates that6-thioguanosine was used as a positive control in place of inosine.“***” indicates questionable activity. “****” indicates that the assaywas sensitive to boiling. “#” indicates that these assays wereterminated by filtering and not by boiling.

EXAMPLE 13

In Vivo Treatment with Bacterial PNP and MeP-dR

The utility of the bacterial PNP and prodrugs such as MeP-dR to inhibitcancer growth in vivo was demonstrated in mice engrafted with tumorsexpressing the bacterial PNP gene. The first step required theproduction of a recombinant retrovirus containing a constitutivelyexpressed bacterial PNP gene, as described above. The bacterial PNPencoding sequence was excised from the SV/PNP plasmid and ligated bystandard techniques into the pLNSX vector. The resulting vector, pLN/PNPused the SV40 early promoter to constitutively direct the bacterialtranscription. This plasmid vector was transfected into the ψ2 packagingcell line. The supernatant collected from these cells 48 hours later wasused to infect additional ψ2 packaging cells. Twenty-four hours later,the cells were replated at a lower density (1:5-1:10) in mediacontaining G418 in order to select for clones containing the retroviralsequences. Several clones were selected and titers of clones determinedby standard techniques. A clone with the highest titer was selected asthe source of recombinant, LN/PNP virus, and used to infect tumor cells.

The murine mammary carcinoma cell line, 16/C, was modified toconstitutively express the bacterial PNP by infection with the LN/PNPvirus. The 16/C cells were plated at a subconfluent density, and theLN/PNP virus contained within the supernatant from the ψ2-producer linewas applied in the presence of polybrene (5 μg/ml) for several hours.The media was changed to normal media for 24 hours, after which thecells were enzymatically detached and plated at a lower density in mediacontaining G418 (1 gm/L) to select infected cells. A polyclonal mixtureof G418 resistant cells, to be referred to here as “16/C-PNP cells”, wasamplified in number for engraftment into mice. Further description ofthe methods for generation of stable PNP expressing tumor cell lines isalso provided below.

Athymic (nude) mice were engrafted with the 16/C-PNP cells. Each mousereceived 2×10⁶ cells subcutaneously (sq) in the left flank on day 1. Theresults are shown in FIG. 4. Control animals (n=4) were maintained undernormal nude mouse conditions that resulted in measurable tumors by day13. The tumors in all of the control mice continued to increase in sizethrough day 29 following engraftment. The early treatment group (n=4)was treated by intraperitoneal (IP) injections of 6-MeP-dR at 100 mg/kg,a dose just below the maximum tolerated dose, each day for the first 4days (days 1-4). One of these mice was sacrificed at day 8 to studytumor histology, and two more died at day 20, from undetermined causes,possibly due to the very high levels of prodrug administered.Importantly, none of the mice had any detectable tumor up to 18 dayspost engraftment. One mouse developed a very small tumor at day 22. Thelate treatment group (n=4) was treated by intraperitoneal injections of6-MeP-dR at 100 mg/kg each day on days 13, 14, and 15 post engraftment.All of the late treatment group had tumors of comparable size to thecontrols on day 13. Unlike the controls, the tumors in the latetreatment group did not increase in size after day 15. All of theseanimals survived for the complete experiment. These results clearly showthat the combination of the bacterial PNP plus prodrug causes areduction in tumor growth in vivo.

EXAMPLE 14

Generation of Stable-cell Lines Expressing E. coli PNP

High level bystander killing of cancer cells in vitro was evaluatedusing stable, PNP expressing cell lines. The E. coli PNP gene was clonedinto the Hind III and Stu I sites of LNSX, a retroviral vector (Milleret al., Biotechniques 7:980-990 (1989)) in which the neomycin resistancegene is LTR-driven, and the SV40 early promoter regulates E. coli PNPexpression. Cloning was accomplished by excising the E. coli PNP genefrom SV-PNP and directionally cloning the fragment into LNSX (Sorscheret al., Gene Ther., 1:233-238 (1994)). The construct was thentransfected using the Lipofectin reagent (Gibco BRL) into an ecotropic3T3-based packaging cell line (ψ2). In order to obtain a higherretroviral titer, supernatants from the initial viral collection wereused to transduce fresh ψ2 cells. Fresh medium and G418 (Gibco BRL) wereadded every 3 days. Producer cells capable of releasing 10⁴-10⁵infectious particles/ml growth medium were obtained, and used totransduce murine melanoma (B16), murine breast carcinoma (16/C), andhuman glioma (D54) cell lines. Three days following addition of virus,transduced cells were selected with G418 as above.

EXAMPLE 15

Cloning of the Human Tyrosinase Promoter Region and Construction ofLuciferase Reporter Vectors

Two polymerase chain reaction primers,(GATCGCTAGCGGGCTG-AAGACAATCTCTCTCTGC (SEQ ID NO. 6) andGATCGCTAGCTTCCTCTA GTCCTCACAA-GGTCT (SEQ ID NO. 7)) were used to definethe 529 base pairs (bp) of the human tyrosinase promoter immediatelyupstream of the start of translation (−451 to +78) and to incorporateNhe I sites (underlined) at both 5′ and 3′ ends of the desired product(Giebel et al., Genomics, 9:435-45 (1991); Kikuc et al., Biochem.Biophys. Acta., 1009:283-6 (1989)). Template DNA was prepared from wholehuman blood as described by Sorscher et al., Lancet, 337:1115-8 (1991).After 30 cycles of amplification, a single PCR product of the predictedsize (553 base pairs) was obtained (94° C.×1 min, denaturation, 50° C.×2minutes annealing, and 72° C.×3 minutes elongation) using I ng template,100 ng of each primer in a 100 μl reaction mixture containing 2.5 unitsTaq polymerase, 200 mM of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 8.3),1.5 mM MgCl₂, and 0.01% gelatin (weight/vol.). This product wasextracted with phenol/chloroform, precipitated with ethanol, digestedwith Nhe 1, and gel purified. A luciferase reporter gene vector lackingany promoter (pGL2 Basic vector, Promega) was cut with Nhe I and theabove PCR product was ligated immediately upstream of the luciferasegene. Recombinants were screened by restriction mapping and a correctlyoriented clone was identified (Tyr-Luc). A plasmid with the tyrosinasepromoter in reverse orientation (Rev-Tyr-Luc), for use as a negativecontrol, was also selected.

EXAMPLE 16

Cancer Cell Lines for Studying Gene Activation by the TyrosinasePromoter

B16 and 16/C are of murine origin and were a gift of Dr. W. Waud,Southern Research Institute, Birmingham, Ala.; all other cell lines areof human derivation. Mel-I (melanoma) was provided by T. Carey,University of Michigan as UMCC-Mel-1. Mel-21 (melanoma) was provided byM. B. Khazaeli, University of Alabama, Birmingham. GP6F2 (prostate) wasa gift of M. Moore, Grady Memorial Hospital, Atlanta, Ga. U-373 and D54(glioma) were provided by Yancey Gillespie, University of Alabama,Birmingham. HeLa (cervical carcinoma), Hep G2 (hepatocellularcarcinoma), and T-84 (colon carcinoma) were obtained from the AmericanType Culture Collection. Mel-1, Mel-21, Hep G2, and HeLa cells werecultured in Earle's minimal essential medium containing Earle's salts,and 1% L-glutamine (Gibco-BRL), with 10% fetal bovine serum and 1%nonessential amino acids. T-84 and GP6F2 cells were cultured in a 1:1mixture of Dulbecco's modified Eagle's medium and nutrient mixture F-12(Ham's ) (Gibco-BRL) with 15 mM HEPES, 1% L-glutamine, and 10% fetalbovine serum. B 16, 16/C and RPMI 8226 cells were cultured in RPMImedium 1640 with 1% L-glutamine (Gibco-BRL) and 10% fetal bovine serum.All cells were cultured at 37° C. with 85% humidity and 5% CO₂.

EXAMPLE 17

Luciferase and X-gal Assays

Each plate was washed three times with PBS and 100 μl of lysis buffer(Luciferase Assay System, Promega) was added to each well of a six-wellplate. After 15 minute incubation at 37° C., the lysate and cell debriswere collected. Forty μl of the lysate was added to 100 μl of luciferaseassay substrate (Promega) in a clear polystyrene 12×75 mm tube,immediately placed in a luminometer (Analytical Luminescence Laboratorymodel 2010) and light production measured for 15 seconds. X-gal stainingfor transfection efficiency using LacZ constructs was as described bySorscher et al., Gene Ther., 1:233-238 (1994).

EXAMPLE 18

Killing and Proliferation Assays

In some studies, cellular toxicity (percentage of dead cells) wasmeasured by LDH release from dying cells (Promega, CytotoxJ 96 kit). Theproliferation assay (living cell number/well) was performed using ameasurement of tetrazonium conversion to formazin during cell growth(Cell TiterJ 96 kit). Since these two assays are designed to studyapproximately 10,000 cells per condition (using 96 well trays),measurements of bystander effects below approximately 1% (100 transducedcells) were effectively limited by difficulty in accurately countingvery small numbers of transduced, viable cells.

EXAMPLE 19

Implantation of Tumor Cells into Mice

Transduced 16/C cells were implanted in mice by subcutaneous injectionof approximately 10⁶ cells harvested from the cultures of stablytransduced 16/C cells described above. The mice were examined visiblyfor tumor growth and those with developing tumors were maintained. Toprepare mice for use in the in vivo experiments, the tumors were removedfrom mice with significant tumor growth and cut into 30-60 mg pieces.One 30-60 mg piece of the tumor was subcutaneously implanted into thesubaxillary region of each female B6C3F1 mouse. The tumors were allowedto develop and mice with tumors of 100 mm³ were used.

For studies conducted with nu/nu mice, cells obtained from stablecultures of transduced cell lines were injected subcutaneously into theright or left flank of the mice. Mice with visible tumor growth wereused for further studies. For the administration of purine prodrug, micewere administered MeP-dR or F-araAMP by IP injection.

EXAMPLE 20

Bystander Killing by Cell Lines Expressing E. coli PNP

Transient E. coli PNP expression in a human colonic carcinoma cell lineis capable of mediating total cell population killing in vitro even whenonly approximately 1% of cells express the E. coli PNP gene (FIG. 1).The growth characteristics of wild type and transduced B16 cells, andwild type and transduced 16c cells were the same in the absence ofMeP-dR. In FIG. 5, a dose of MeP-dR (20 μg/ml) which is not toxic tountransduced B16 melanoma or 16/C breast cancer cells was added to mixedcultures containing an increasing population of transduced E. coli PNPexpressing cells. Effects on both cell proliferation and cell survivalwere evaluated in the presence or absence of MeP-dR. In theseexperiments, concentrations of MeP-dR which had no effect onuntransduced (wild type) B16 (FIG. 5A) or 16/C (FIG. 5B) tumor cellscompletely eliminated cell proliferation even when as few as 2% of cellsin culture expressed the E. coli PNP gene. Based upon an LDH releaseassay, total population cell killing required that 10% of B16 cells and#1% of 16/C cells expressed the PNP gene. When E. coli PNP activity inthe transduced B16 and 16/C cells was assayed by direct enzymaticmeasurement using cell free extracts, the activity measured intransduced 16/C cells was approximately 4 fold higher than in B16 cells.(16/C:10.7 nmoles MeP-dR converted/mg cell protein/hr (n=6); B16: 2.4nmoles MeP-dR converted/mg cell protein/hour (n=2); background activityin non-transduced 16/C and B16 cells was 0 (n=4 measurements for eachcell line)).

EXAMPLE 21

Killing of Malignant Cells in vivo: Growth of 16/C Mouse BreastCarcinoma in B6C3F1 Mice

Six mice (B6C3F1) per group with established wild type 16/C tumors weretreated with an aqueous control solution, MeP-dR (100 mg/kg IP qd×3d) or2-fluoro-arabinofuranosyladenine monophosphate (F-araAMP) (100 mg/kg IP,5 id×3d). The wild type tumors grew rapidly in the presence or absenceof either of the prodrugs. In addition, there was no statisticallysignificant delay in tumor growth attributable to either prodrug. (TableIII, Wild-type 16/C treatment). This demonstrated that the prodrug wasnot toxic to the mice at the doses given and had no effect on non-PNPexpressing tumor cells.

Six mice per group with established PNP-transduced 16/C tumors weretreated with aqueous control solution, MeP-dR or F-ara AMP, as above.Control solution treated tumors grew rapidly, comparable to the rate ofgrowth observed with the wild type tumors. Complete tumor regression wasobserved in three of six in the MeP-dR treated group. In addition, astatistically significant delay in the time necessary for three tumordoublings was noted for the MeP-dR treated group (p<0.01) and theF-araAMP treated group (p<0.01). (Table III, 16/C-PNP treatment.)

TABLE III Effect of MeP-dR and F-araAMP on the growth of wild-type 16/Ctumors and 16/C tumors transduced with the E. coli PNP gene (B6C3F1)Complete Days for tumor Dose/day** Regressions/ Nonspecific to double 3Day delay Treatment (mg/kg) Total Deaths/Total times * mean/SD(Treated-control) Wild-type 16/C Vehicle — 0/6 — 6.2 ∀ 3.7   — MeP-dR100 0/6 0/6   8.6 ∀ 0.7   2.4 F-araAMP 500 0/6 0/6   8.9 ∀ 2.0   2.716/C-PNP Vehicle — 0/6 — 8.8 ∀ 1.1   — MeP-dR 100 3/6 2/6**** 14.2 ∀3.2*** 5.4 F-araAMP 500 0/6   0/6 12.1 + 1.6*** 3.3 * refers to the mean∀ the standard deviation of the days to 3 doublings of the tumors thatcontinued to grow in the presence of drug, and does not include thetumors that completely regressed. **refers to mice (B6C3FI) implanted(SC) with wild-type 16/C tumors or E. coli PNP-transduced 16/C tumors(16/C PNP). Three days post implantation, when tumors had grown toapproximately 100 mg, the animals were treated (IP) with vehicle, 100mg/kg of MeP-dR once a day for three days, or 100 mg/kg of F-ara-AMPfive times a day (2 hour # intervals) for three days. ***refers to asignificant difference from the growth rate of 16/C-PNP tumors inanimals treated with vehicle, p < 0.01, Student's t test. The growthrate of wild-type 16/C tumors treated with MeP-dR and F-araAMP was notsignificantly different from vehicle-treated tumors, and the growth ofvehicle-treated wild-type 16/C tumors was not # significantly differentfrom the growth rate of vehicle-treated 16/C-PNP tumors. ****in the invivo experiments described, MeP-dR dosages were given at just below themaximum tolerated dosage. As expected, the near lethal dosage of MeP-dRresulted in sporadic animal death (occurred in some animals 1-2 weeksfollowing complete or substantial tumor regression).

EXAMPLE 22

Immunological Clearance of Tumors

To demonstrate that the efficacy shown above was not due to immuneresponse and clearance of PNP-expressing tumors, immune deficient mice(nu/nu) were studied using a similar protocol. Four to five nude (nu/nu)mice per group were inoculated with wild type murine breast carcinomacells (16/C cell line), or PNP transduced 16/C cells. Mice withestablished tumors (approximately 100 mm³) were treated with MeP-dR (100mg/kg/d IP×3 d) or F-araAMP (100 mg/kg/IP 3 id×3 d).

Wild type tumors grew rapidly following either vehicle or prodrugadministration (FIG. 6). Animals with PNP-transduced tumors which weretreated with F-araAMP for three days demonstrated evidence of growthdelay for at least ten days, FIG. 7. Animals treated with MeP-dR showedsubstantial antitumor effects whether treated at a time when tumors wereestablished (days 13-15) or immediately following tumor cellsinoculations (days 1-4), FIG. 4.

EXAMPLE 23

In vivo Activity of MeP-dR Against Human Glioma Transduced with E. coliPNP

Female athymic nude mice (nu/nu) were implanted sc with 2×10⁷ cells ofeither D54 parental tumor cells (D54-wt) or D54 tumor cells that hadbeen transduced with the E. coli PNP (D54-PNP). After the tumors hadgrown to approximately 150 mg, they were treated ip with either vehicleor 67 mg/kg of MeP-dR (IP) once a day for 3 days (days 6, 7 and 8 afterimplantation). The tumor sizes were measured twice a week aftertreatment.

There were 6 of 6 complete regressions in mice with the D54-PNP tumorsthat were treated with MeP-dR (Table IV). Four of these animals had nodetectable tumors at the termination of the experiment. MeP-dR hadlittle effect on the D54-wt tumors. There was little or no loss ofweight in the animals that were treated with 67 mg/kg of MeP-dR,regardless of tumor implanted. Animals were followed for a total of 65days. No treated animals died in these experiments (FIGS. 8 and 9).

TABLE IV Effect of MeP-dR on the growth of wild-type D54 tumors and D54tumors transduced with the E. coli PNP gene. Regressions NonspecificDoubling Days Delay Tumor-free Treatment Complete Partial Deaths/Totaltime (T-C) Survival Wild-type D54 Vehicle — — — 14 — 0/10 MeP-dR (67)0/6 0/6 1/6 21  7 0/6  D54-PNP Vehicle — — — 17 — 0/10 MeP-dR (67) 6/60/6 0/6 >56   >39   4/6  **, Mice (NCr-nu) were implanted (SC) withwild-type D54 tumors or E. coli PNP-transduced 16/C tumors (D54-PNP).When tumors had grown to approximately 100 mg, the animals were treated(IP) with vehicle or 67 mg/kg of MeP-dR once a day for three days.

A confirmation experiment was set up exactly as described above, exceptthat animals were treated with two doses of MeP-dR (45 and 67 mg/kg)(FIGS. 10 and 11). The results of this experiment were similar (TableV). There were 8 of 10 complete regressions in mice bearing the D54-PNPtumors that were treated with 67 mg/kg of MeP-dR. In 4 mice the tumorssubsequently returned and grew. There were still 4 of 10 tumor-freesurvivors 60 days after the treatment had stopped. Treatment with 45mg/kg MeP-dR also had a marked affect on mice bearing the D54-PNPtumors. There were 2 of 10 complete regressions and 3 partial responses.There were no tumor-free survivors in animals bearing the D54-PNP tumorthat were treated with 45 mg/kg MeP-dR. Again, there were no partial ofcomplete remissions in animals bearing the D54 wild-type tumors treatedwith either 45 or 67 mg/kg of MeP-dR. The delay in the time required todouble twice due to treatment with MeP-dR was 5 to 6 days in thenon-transduced tumors and greater than 24 days in transduced tumors. Inthis experiment, the growth rate of the D54-PNP tumors was considerablyslower than it was in the first experiment. There was no change in thegrowth rate of the D54-wt tumors. The reason for the slow growth rate ofthe D54-PNP tumors in this experiment is not known. The Figures shown(FIGS. 8-11) only describe the growth of tumors that did not showcomplete regression. (In other words, if a tumor was too small tomeasure, it was not included in the average size). This means that theoverall tumor regressions in the D54 PNP group are actually much morepronounced than they appear in FIGS. 8-11.

TABLE V Effect of MeP-dR on the growth of wild-type D54 tumors and D54tumors transduced with the E. coli PNP gene. Regressions NonspecificDoubling Days Delay Tumor-free Treatment Complete Partial Deaths/Totaltime (T-C) Survival Wild-type D54 Vehicle — — — 12 — 0/10 MeP-dR (45)0/10 0/10 0/10 17  5 0/10 MeP-dR (67) 0/10 0/10 0/10 18  6 0/10 D54-PNPVehicle — — — 30 — 0/10 MeP-dR (45) 2/10 3/10 0/10 >54   >24   0/10MeP-dR (67) 8/10 1/10 1/10 >55   >25   4/10 **, Mice (NCr-nu) wereimplanted (SC) with wild-type D54 tumors or E. coli PNP-transduced 16/Ctumors (D54-PNP). When tumors had grown to approximately 100 mg, theanimals were treated (IP) with vehicle, 45 or 67 mg/kg of MeP-dR once aday for three days.

These results show that it is possible to cure animals that generate MePfrom MeP-dR at the site of the tumor without killing the animal. This isimportant because MeP is a toxic agent and these results alleviate theconcern that doses sufficient to destroy the tumor would release anamount of MeP into the body that would kill the animal. Therefore, theseresults indicate that the MeP released from PNP-expressing tumors isdiluted by body fluids to concentrations below a toxic level. The genetherapy methodology of the present invention, therefore, offers a newway to generate highly toxic chemotherapeutic drugs within a growingtumor, in such a way as to completely eliminate the tumor without undueweight loss or other apparent toxicity. Taken together, the presentinvention demonstrates the usefulness of a new class of antitumor agentsto treat of breast, melanoma, glioma, and other refractory solid tumortypes in vivo.

Other additional in vivo experiments indicate that: (1) very largepre-existing tumors (approximately 1 gram in size) transduced with E.coli PNP show impressive regression when treated with 67 mg/kg of MeP-dR(ip, qd×3 d); (2) F-araAMP, a clinical useful drug in human, leads to invivo regression of PNP transduced tumors in mice; (3)2-F-2′-deoxyadenosine can be given to mice in doses similar to MeP-dRwithout toxicity, and mediates strong anti-tumor effects, equivalent toor above those seen with MeP-dR. This suggests that2-F-2′-deoxyadenosine should be a useful prodrug in vivo, since theliberated toxin, 2-F-Ade, is 10 to 100 fold more toxic than MeP.

EXAMPLE 24

Other Prodrugs

In addition to MeP-dR, two other prodrugs suitable for E. coli PNPactivation in tumor cells can be applied to the methodology of thepresent invention. These prodrugs are F-araA and 2-F-2′deoxyadenosine.Both show high level killing of PNP-transduced tumor cells in vitro. Adose of 2-F-2′-deoxyadenosine (100 μM) was defined in the presence of 1mM deoxycytidine that kills cells transduced with the E. coli PNP evenwhen as few as 1% of the tumor cells express the gene. As desired, thisdose had no effect on control untransduced, tumor cells. A dose ofF-araA (500 ng/ml) also was identified that specifically killedtransduced, but not untransduced, tumor cells.

In addition, 21 purine nucleoside analogs were evaluated as substratesfor E. coli PNP by an independent protocol (Table II). These resultshave identified 5 compounds as possible prodrugs in this strategy;MeP-dR, 2-F-2′-deoxyadenosine, 1 deaza-2-amino-6-Cl-purine-riboside,7-ribosyl-3-deazaguanine, and 7-ribosyl-6-mercaptopurine. All of thesecompounds have the following characteristics: the nucleoside analog isrelatively nontoxic when compared to the base of which it is composed,the nucleosides are good substrates for the E. coli PNP, and they arepoor substrates for the human PNP. Three agents that were poorly cleavedby the E. coli PNP but were not cleaved by the human enzyme were5′-amino-5′-deoxyadenosine, 2-F-arabinofuranosyl-adenine, andα-adenosine. Compounds that were poor substrates for both the human andE. coli PNP were also identified. These compounds include xylosylmethylpurine, 2′,3′-dideoxyadenosine, 3′-deoxyadenosine, 5′-carboxamideof adenosine, and the isopropylidine of the 5′-carboxamide of adenosine.

Kinetic constants for the cleavage of inosine, MeP-dR, F-dAdo, andF-araA by E. coli PNP were determined from enzymes isolated from eithertransduced human cells or E. coli cell pellets (Table VI). The resultsof these experiments indicated that there were little or no differencesbetween the prokaryotic E. coli PNP enzyme in bacteria and after tumorcell expression of the recombinant enzyme. In addition, it was clearthat inosine, MeP-dR, and F-dAdo were similar as substrates forrecombinant and natural E. coli PNP. F-araA was poorly cleaved by E.coli PNP with K_(m) of 543 μM and V_(max) of 1.9 nmole/mg/minute.

TABLE VI Kinetic constants of MeP-dR, F-dAdo, and F-araA with E. coliPNP Sub. Source K_(m)(μM) V_(max) V_(max)/K_(m) Inosine Bacteria 46 1322.9 D54 cells — — — MeP-dR Bacteria 68 251 3.7 D54 cells 107 5.4 0.050F-dAdo Bacteria 44 190 4.3 D54 cells 38 2.1 0.056 F-araA Bacteria 5431.9 0.0034 D54 cells 510 0.023 0.000043

EXAMPLE 25

Recombinant E. coli that Express 100-Fold More PNP Activity ThanWild-Type E. coli and Use of this Bacterium to Deliver E. coli PNP toTumor Cells

Previous studies showed that MeP-dR, F-araAMP, and F-dAdo have goodactivity against D54 glioma tumors expressing the E. coli PNP gene.MeP-dR, F-araAMP, and F-dAdo were less active against tumors composed ofmixtures of wild-type tumor cells and transduced tumor cells at a ratioof 80 to 20, respectively. This result indicated that increasedexpression of E. coli PNP in the tumor cell may be necessary todemonstrate in vivo bystander activity with these three compounds. Theamount of expression of E. coli PNP activity in the transduced D54tumors cells was between 200 to 300 nmoles of MeP-dR cleaved per mgprotein per hour.

In an effort to increase the amount of E. coli PNP expressed in tumorcells and to develop a vector to realistically deliver E. coli PNP totumors in animals, E. coli was transformed with the E. coli PNP gene(SEQ ID No:5) and created a recombinant E. coli that expressed very highlevels of E. coli PNP, approximately 1,000,000 nmoles of MeP-dR cleavedper mg protein per hour. In order to accomplish this, a plasmid capableof mediating high level expression of E. coli PNP was constructed byexcising the E. coli PNP (SEQ ID No:5) from a transfer vector (pTM-1PNP) by a double restriction enzyme digestion with Nco I and Xho I. (seeFIG. 12). pTRC His B (Invitrogen, Carlsbad, Calif.) was digested withNco I and Xho I, and the E. coli PNP fragments described above weredirectionally ligated into pTRC so as to initiate E. coli PNPtranslation from the first methionine. The ligation reaction was used totransform competent E. coli by standard techniques. In this case, theDH5% strain was used, but other strains of E. coli or other bacteriacould be used for the same purpose. Recombinants were selected onampicillin, and correct orientation and integration of the insert wasverified by restriction mapping. The plasmid is designed to allowfurther induction of E. coli PNP activity after treatment withisopropyl-%-D-thio-galactopyranoside (IPTG), and in some experimentsthis induction was verified as additional evidence of the predictedbehavior of the recombinant plasmid. FIG. 13 shows a protein band thatis present in the recombinant E. coli (Lanes 10, 11, 15) that isinducible with IPTG (Lanes 15′, 11′, and 10′). No protein was detectedin the wild-type strains at this position, or in strains expressing aninducible control protein (WT NBD2 and WT NBD2′). The amount of E. coliPNP in wild-type bacteria was approximately 10,000 nmoles of MeP-dRcleaved per mg protein per hour. Therefore, this recombinant E. coli had100-fold more E. coli PNP activity than wild-type cells.

Tumors in the flanks of mice were injected with this recombinant E.coli, and the activity of E. coli PNP in the tumors was determinedthirty minutes and forty-eight hours after injection of bacteria. Afterthirty minutes the amount of E. coli PNP activity in the tumors wasapproximately 100,000 nmoles of MeP-dR cleaved per mg protein per hour,whereas at forty-eight hours the activity in the tumors had increasedmodestly to approximately 200,000 nmoles of MeP-dR cleaved per mgprotein per hour (see Table VII). These results indicated thatapproximately 1000 times more E. coli PNP could be delivered to tumorcells than was present in the D54-PNP tumors. This result verifies thathuman patients could be treated by inoculating their tumors with thisrecombinant bacteria.

TABLE VII E. coli PNP activity in tumors injected with recombinant E.coli Sample 30 minutes 40 hours 1-1 0.1 ml/tumor 100,000 >223,000-2 >178,000 >133,000 -3 78,000 >212,000 2-1 0.2ml/tumor >223,000 >227,000 -2 >184,000 >222,000-3 >179,000 >292,000 >indicates that there was considerable cleavage ofMeP-dR (>40%) at the earliest time measured (fifteen minutes). There waslittle or no cleavage at zero (0) time. The specific activity wasdetermined from this number which was not in the linear portion of theactivity curve. Therefore, these numbers are a slight underestimate ofthe true activity.

EXAMPLE 26

Treatment of Lewis Lung Tumors with E. coli and MeP-dR

Subcutaneous Lewis Lung tumors on the flanks of mice (approximately 300mg) were injected with E. coli bacteria transfected with E. coli purinenucleoside phosphorylase gene (plasmid pTRCPNP) containing SEQ ID No: 5.Mice were treated with 0, 16.8, 33.5, or 67 mg/kg of MeP-dR once a dayfor three days, and the tumor size was monitored over the following 18days. The control consisted of saline/Tween 80. This initial experimentwas designed for two purposes. First, to determine whether E. coliover-expressing the PNP gene could be given in combination with MeP-dRand without undue toxicity. Second, to evaluate anti-tumor effects inthis particular animal model and strain of mouse. E. coli PNP activitywas measured thirty minutes and forty-eight hours after injection ofbacteria in representative Lewis Lung tumors injected with bacteria butnot treated with MeP-dR. The PNP activity was 16,000 and 28,000 nmolesof MeP-dR cleaved per mg protein per hour at 0.5 and forty-eight hours,respectively (each number is the average of two determinations). Theanti-tumor results (shown in FIG. 14) indicated that treatment with E.coli bacteria that express E. coli PNP activity plus MeP-dR slowed thegrowth of these fast growing tumors. In this experiment, treatment with33.5 mg/kg of MeP-dR delayed tumor growth by approximately 42% withouthost toxicity. Treatment with a higher dose of MeP-dR (67 mg/kg, IPQd×3d) was toxic, with several deaths due to the combined therapy.Nevertheless, this treatment arm was informative, since anti-tumoreffects were again observed. These results indicated that E. colibacteria could deliver significant amounts of E. coli PNP to tumor cellsin an animal and that this enzyme could activate MeP-dR resulting in anantitumor response.

EXAMPLE 27

Injection of Methyl Purine (MeP) into Established Human PancreaticTumors

CF PAC (human pancreatic adenocarcinoma) cells were grown in T75 flasks,trypsinized, washed with PBS and then inoculated subcutaneously intofemale SCID mice at approximately 2×10⁷ cells per animal. The tumorswere allowed to grow for several days until they reached the size ofapproximately 80-100 millimeters. At this point, groups of five animalswere treated either with vehicle (PBS), PBS containing methyl purine(1.67 mg/kg) or PBS with a higher dose of methyl purine (5.0 mg/kg).Drug or vehicle was injected intratumorally with 3-4 needle passes.Animals were treated with either vehicle or drug one time each day forthree days. The growth of the tumors was followed. Measurements in twodimension were used to approximate the overall tumor size. Tumorstreated at the higher dose of MeP were completely arrested in terms oftheir growth, while the lower dose of MeP led to an intermediate effectupon tumor growth. The results (see FIGS. 15 and 16) indicated that MePhas a very potent anti-tumor effect when injected directly into tumorsand further support the notion that methyl purine and derivativesthereof is an effective anti-tumor agent, either after direct tumorinoculation or when generated within a tumor by virtue of expression ofthe E. coli PNP gene. The results also suggest that direct inter-turmoalinjection of cytotoxic purine analogs or other cytotoxic drugs canelicit profound anti-tumor effects in vivo. For example, purine analogswhich are known to have strong cytotoxic activity such as2-fluoroadenine, 2-fluoroadenosine, 9-[ribosyl]-6-methylpurine,2-amino-6-chloro-1-deazapurine, 3-deazaguanine, 6-thioguanine, and6-mercaptopurine can be directly administered to a tumor to causeregression and/or inhibit tumor growth.

Summary

The following data summarizes in vitro and in vivo experiments in whichthe efficacy of the claimed delivery vehicles and methods were furtherdemonstrated. Experiments to show the killing of cancer cells in vitroused mixed populations of PNP expressing and nonexpressing cells. Theresults demonstrated that a small population of PNP expressing cells canfacilitate the death of large numbers of surrounding, non-PNP expressingcells. In vivo efficacy was demonstrated by implanting into micetransduced and nontransduced tumor cells. Tumor size decreased in miceimplanted with PNP-transduced tumor cells upon the administration of aprodrug purine analog. The results indicate that the claimed methods areapplicable to the treatment of mammalian malignant disorders.

The results provided were generated using art recognized in vivo and invitro models of mammalian malignancy. These results demonstrate that:(1) a small number of PNP expressing tumor cells can facilitate in thekilling of surrounding, non-PNP expressing cells, (2) PNP expression canbe controlled in a tissue specific fashion, (3) the claimed therapeuticmethod works with a variety of tumor types in art recognized models ofmammalian malignancy, and (4) that purine analogs such as methyl purinecan be used to inhibit tumor growth.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentmethods, procedures, treatments, molecules, and specific compoundsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

1. A method of killing replicating or non-replicating, targetedmammalian cells and bystander cells, comprising the steps of: (a)delivering directly to the targeted mammalian cells a purine cleavageenzyme; and (b) contacting the targeted cells with an effective amountof a substrate for the purine cleavage enzyme, wherein the substrate isnon-toxic to mammalian cells and is cleaved by the cleavage enzyme toyield a purine base which is toxic to the targeted mammalian cells andbystander cells, to kill the mammalian cells contacted with the cleavageenzyme and the bystander cells.
 2. The method of claim 1, wherein thesubstrate is selected from the group consisting of9-(β-D-2-deoxyerythropentofuranosyl)-6-methylpurine,2-amino-6-chloro-1-deazapurine riboside, 7-ribosyl-3-deazaguanine,arabinofuranosyl-2-fluoroadenine, 2-fluoro-2′-deoxyadenosine,2-fluoro-5′-deoxyadenosine, 2-chloro-2′-deoxy-adenosine,5′-amino-5′-deoxy-adenosine, α-adenosine, MeP-2′,3′-dideoxyriboside,2-F-2′, 3′-dideoxyadenosine, MeP-3′-deoxyriboside,2-F-3′-deoxyadenosine, 2-F-adenine-6′-deoxy-β-D-allofuranoside,2-F-adenine-α-L-lyxofuranoside, MeP-6′-deoxy-β-D-allofuranoside,MeP-α-L-lyxofuranoside, 2-F-adenine-6′-deoxy-α-L-talofuranoside,MeP-6′-deoxy-α-L-talofuranoside and 7-ribosyl-thioguanine.
 3. The methodaccording to claim 1, wherein the enzyme is targeted to the cells byconjugating the enzyme to an antibody.
 4. The method according to claim1, wherein the enzyme is selected from the group consisting of a naturalnon-human purine nucleoside phosphorylase (PNP), a natural non-humanhydrolase, a natural human purine nucleoside phosphorylase (PNP), and anatural human hydrolase.
 5. The method according to claim 1, wherein thetargeted cells are selected from the group consisting of: tumor cellsand virally infected cells.